Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Tissue distribution of bovid herpesvirus-4 in inoculated rabbits and its detection by DNA hybridization and polymerase chain reaction

Summary A DNA hybridization technique, using the polyrepetitiveEcoRI L-fragment of bovid herpesvirus (BHV-4) as a probe, was developed to determine virus distribution in the tissues of BHV-4-infected pregnant rabbits. The cloned fragment did not react with the DNA of rabbits or of other herpesviruses, e.g., infectious bovine rhinotracheitis, bovine herpes virus mammillitis, and pseudorabies viruses. The detection limit was 10^–13g of DNA or approximately 600 genome equivalents of viral DNA, which indicates a level of sensitivity of one viral genome per 500 cells in our assay. Using conventional cell culture techniques, the virus was isolated from only one of fifteen infected rabbits and a few aborted fetuses. However, when organ culture or dot blot hybridization was used, BHV-4 was detected in all rabbits and their fetuses. Viral DNA was detected by DNA hybridization in spleen, ovary, uterus, lung, liver, salivary gland, lymph node, and placentome of adult rabbits and in a composite of fetal tissues. When polymerase chain reaction (PCR) was used, the virus was detected in several organs (including the nervous tissues) that were found negative by other techniques. These results indicate that blot hybridization and PCR are more sensitive than conventional techniques for studying the pathogenesis of BHV-4 in animals. The data obtained by these methods suggest that BHV-4 may be maintained in infected rabbits in a latent state in a variety of tissues including the nervous system.     

The impact of telomere erosion on memory CD8+ T cells in patients with X-linked lymphoproliferative syndrome

Patients with X-linked lymphoproliferative syndrome (XLP) experience excessive T cell proliferation after primary Epstein-Barr virus (EBV) infection, due to mutations in the signalling lymphocyte activation molecule (SLAM) associated protein (SAP) molecule. We examined the impact of dysfunctional proliferative control on the extent of CD8+ T cell differentiation in XLP patients who recovered from primary EBV infection. Although these young patients have normal numbers of lytic and latent EBV-epitope-specific CD8+ T cells, they were extremely differentiated as defined by loss of CCR7 and CD27, low telomerase activity and very short telomeres. This was not a direct effect arising from the loss of SAP, but was due to excessive T cell stimulation due to this defect. Thus, transduction of XLP CD8+ T cells with the catalytic component of telomerase (hTERT), but not SAP, prevented telomere loss and considerably extended proliferative lifespan in vitro. These results indicate that excessive proliferation in CD8+ T cells in XLP patients may lead to end-stage differentiation and loss of functional EBV-specific CD8+ T cells through replicative senescence. This may contribute to the defective immunity found in XLP patients who survive acute EBV infection who develop EBV-related B cell lymphomas before the fourth decade of life.     

Reconstitution of T-cell repertoire after autologous stem cell transplantation: influence of CD34 selection and cytomegalovirus infection.

The period of immunodeficiency following autologous hematopoietic stem cell transplantation is characterized by transient expansions of CD8+CD45RO+CD57+ T lymphocytes, displaying markers of an activated phenotype. Most evidence suggests that this early reconstitution results from proliferation of mature T cells that have survived conditioning or were transferred with the graft. Although homeostatic mechanisms are thought to act in maintaining total T-cell numbers, the degree to which antigen-driven expansions contribute and the nature of the stimulating antigens remain unclear. CD34 selection of stem cell grafts reduces the available T-cell pool, potentially delaying immune reconstitution and resulting in increased infective complications. In the allogeneic transplantation setting, lymphopenia has been associated with cytomegalovirus (CMV) infection risk and, if persistent, with adverse outcome. We prospectively studied patients undergoing CD34-selected (n = 13) or unselected (n = 13) autologous hematopoeitic stem cell transplantation for immune reconstitution and CMV infection. No significant differences were demonstrated between graft types with respect to lymphocyte subset recovery, T-cell receptor beta-chain variable region spectratype diversity, or CMV DNA detection rates (45% versus 40%). CMV infection was associated with a trend toward higher rather than lower CD8+ counts at 6 weeks posttransplantation (P =.08) that became significant by 3 months (P=.007), and that was associated with decreased T-cell receptor beta-chain variable region spectratype diversity (P =.01). CMV-specific HLA-tetramer analysis demonstrated transient expansions with CDR3 lengths corresponding to those of some of the major posttransplantation T-cell expansions demonstrated by spectratype analysis suggesting that CMV-specific T cells contribute to the pattern of immune reconstitution.     

Anterior segment findings in AIDS patients with cytomegalovirus retinitis

Background: Anterior segment findings in AIDS patients presenting with cytomegalovirus (CMV) retinitis have not been specifically addressed in the American literature. Methods: Our study evaluated 21 AIDS patients with CMV retinitis. Results: Nineteen (90%) of these patients exhibited corneal endothelial deposits concurrent with CMV retinitis. The endothelial deposits were microscopic, opaque, linear flecks arranged in a reticular-like fashion. Of 42 eyes evaluated, 32 (76%) demonstrated active CMV retinitis. Corneal endothelial deposits were noted in 26 (81%) of the 32 eyes with retinitis. These corneal endothelial deposits were absent in the eyes which did not have CMV retinitis. Conclusion: Meticulous examination of the retina of an HIV-positive or AIDS patient who presents with reticularly arranged, linear, flecked corneal endothelial deposits should be performed to ensure that the diagnosis of CMV retinitis can be ruled out.     

Phenotypic and genotypic characterization of enteroaggregative Escherichia coli isolates from pediatric population in Pakistan

Enteroaggregative Escherichia coli (EAEC) are a leading cause of diarrhea among children. The objective of this study was to define the frequency of EAEC among diarrheal children from flood‐affected areas as well as sporadic cases, determine multidrug resistance, and evaluation of virulence using an in vivo model of pathogenesis. Stool samples were collected from 225 diarrheal children from 2010 to 2011 from flood‐affected areas as well as from sporadic cases in Pakistan. Identified EAEC isolates were characterized by phylogrouping, antibiotic resistance patterns including the extended‐spectrum beta lactamase spectrum, single nucleotide polymorphism detection in gyrA and parC, and virulence potential using wax worm, G. mellonella. A total of 35 (12.5%) confirmed EAEC isolates were identified among 225 E. coli isolates. EAEC isolates displayed high resistance to tetracycline, ampicillin, and cefaclor. A total of 34.28% were ESBL positive. Single nucleotide polymorphism detection revealed 37.14% and 68.57% isolates were positive for SNPs in gyrA (A₆₆₀‐T₆₆₀) and parC (C₃₃₀‐T₃₃₀), respectively. Phylogrouping revealed that B2 phylogroup was more prevalent among all EAEC isolates tested followed by D, A, B1, and non‐typeable (NT). Infection of G. mellonella with EAEC showed that killing infective dose was 100% higher than E. coli DH5 alpha control. EAEC are prevalent among Pakistani children with diarrhea, they are highly resistant to antibiotics, and predominantly fall into B2 phylogroup. Epidemiologic surveillance of EAEC and other E. coli pathotypes is critical to assess not only the role of these pathogens in diarrheal disease but also to determine the extent of multidrug resistance among the population.     

The John Kay Williams Gold Medal of the Royal College of Surgeons of England and Glasgow 2015

This paper describes the clinical treatment of two cases presented by the recipient of the 2015 Bi-collegiate Membership in Orthodontics John Kay Williams Gold Medal of the Royal College of Surgeons of England and Glasgow. The first case describes the management of an 11-year-old male with a class II Division 1 malocclusion with a 9?mm overjet complicated by an upper anterior odontome and moderate upper arch crowding. The second case describes a 14-year-old female with a class III malocclusion with a reverse overjet complicated by moderate lower arch crowding and previously removed upper permanent canines.