Survival of Neisseria meningitidis outside of the host: environmental effects and differences among strains.

Neisseria meningitidis is a gram-negative bacterium that lives as a commensal in the human nasopharynx. Meningococci are generally non-invasive, but can invade the nasopharyngeal epithelia and enter the bloodstream causing life-threatening illnesses. It is generally thought that meningococci do not survive for long outside the host, and that transmission requires relatively close contact between hosts. There are some reports, however, that meningococci can survive drying on surfaces, including glass, plastic and cloth. Our examination of N. meningitidis strains dried on glass showed differences in survival of isolates belonging to serogroups B, C and W135, including persistence of Cuban, New Zealand, and Norwegian epidemic strains up to 8 days, depending on temperature and humidity. Survival of a New Zealand epidemic strain isolate NZ98/254 under ambient conditions in the laboratory was greatest in winter suggesting that environmental factors impacted survival. For most isolates, including NZ98/254, survival under controlled conditions at 30 °C was greater at 22% than 30% relative humidity. There were also some differences in survival between carriage and invasive strains. The results suggest that N. meningitidis could be transmitted through contact with surfaces outside the host, potentially including contact through shared drinking vessels.

Widespread correction of central nervous system disease after intracranial gene therapy in a feline model of Sandhoff disease.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-ß-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

Molecular modeling and simulation studies of SELEX-derived high-affinity DNA aptamers to the Ebola virus nucleoprotein.

Ebola viral disease (EVD) is a highly infectious and potentially fatal illness with a case fatality rate ranging from 25% to 90%. To effectively control its spread, there is a need for rapid, reliable and lowcost point-of-care (P OC) diagnostic tests. While various EVD diagnostic tests exist, few are P OC tests, and many are not cost-effective. The use of antibodies in these tests has limitations, prompting the exploration of aptamers as potential alternatives. Various proteins from the Ebola virus (EBOV) proteome, including EBOV nucleoprotein (NP), are considered viable targets for diagnostic assays. A previous study identified three aptamers (Apt1. Apt2 and Apt3) with high affinity for EBOV NP using systemic evolution of ligands by exponential enrichment (SELEX). This study aimed to employ in silico methods, such as Phyre2, RNAfold, RNAComposer, HADDOCK and GROMACS, to model the structures of EBOV NP and the aptamers, and to investigate their binding. The in silico analysis revealed successful binding of all the three aptamers to EBOV NP, with a suggested ranking of Apt1 > Apt2 > Apt3 based on binding affinity. Microscale thermophoresis (MST) analysis confirmed the binding, providing dissociation constants of 25 ± 2.84, 56 ± 2.76 and 140 ±3.69 nM for Apt1, Apt2 and Apt3, respectively. The study shows that the findings of the in silico analysis was in agreement with the MST analysis. Inclusion of these in silico approaches in diagnostic assay development can expedite the selection of candidate aptamers, potentially overcoming challenges associated with aptamer application in diagnostics.Communicated by Ramaswamy H. Sarma.

Observations about declining fertility in a feline breeding colony.

Feline breeding colonies are important to the feline industry by preserving traits desirable for a particular breed or in research settings by maintaining medically valuable genetic traits. As breeding females age, their reproductive efficiency declines. The objective of this study was to determine the most common causes of infertility in breeding females that were producing fewer kittens. Knowing the cause and average age of infertility would allow management decisions to be made for the betterment of the colony. The medical records of 70 queens retired from breeding from a single research colony were examined for litter size and number, fertility over their lifespan, and age and reason for removal from breeding stock. Sections of uterus and ovaries were evaluated using gross and histopathological examination for a subset of these queens (46). The data suggests that mature, continuously breeding female cats may show signs of reduced fertility (infertile matings or reduced litter size) as early as 3 years of age and may be a result of undiagnosed Cystic Endometrial Hyperplasia (CEH), endometritis, pyometra and/or ovarian cysts. Evaluation of breeding queens should include periodic ultrasounds to monitor for ovarian cysts and evidence of CEH. Retiring animals from breeding once signs of infertility become apparent is recommended.

In vivo administration of histoincompatible lymphocytes leads to rapid functional deletion of cytotoxic T lymphocyte precursors.

It is well established that a single intravenous injection of F1 lymphocytes can rapidly and specifically reduce the ability of a parental recipient to generate CTL against donor alloantigens in a subsequent MLR. By fluorescently labeling the injected cells, we have been able to identify, and if desired, remove them in cell suspensions prepared from recipient spleen and lymph node. The injected cells, whether F1 or syngeneic, appeared to form part of the normal recirculating pool. Removal of injected F1 cells from responder lymph node or spleen cell suspensions had no effect on the response reduction observed in the 5-d in vitro MLR (typically 80% reduction for responder cells taken 2 d after injection of F1 cells). When the frequency of CTL precursors (CTLp) was measured by limiting dilution, it was reduced to the same degree as the MLR response, implying that response reduction is due to a reduction in the number of activatable CTL in the responder cell suspension. An equal mixture of responder cells from treated (i.e., F1 injected) and control mice gave a measured CTLp frequency equivalent to the average of the separate frequencies, implying the absence of suppressor cells active in vitro. Labeled F1 cells recovered from a first recipient could be used to induce response reduction in a second recipient. The results are discussed in terms of APCs that functionally delete rather than stimulate CTLp that recognize them (i.e., a "veto mechanism"). These experiments appear to rule out a role for in vivo-induced suppressor cells up to 8 d after injection of semiallogeneic cells but do not address the question of whether they are induced at later times.

Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.

Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes.

The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.

Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

Complement-fixing islet cell antibodies in the spontaneously diabetic BB rat.

Fourteen rats of the spontaneously diabetic BB line were bled from the retroorbital sinus approximately every 10 days. Sera taken from an early age up to 20 days after the onset of overt diabetes were assayed for complement-fixing antibodies against antigens of the surface of islet cells (CFA). Dispersed islet cells from normal Wistar rats prelabeled with 3H-leucine were used as targets. Target cells in suspension were incubated with heat-inactivated rat sera and then, after washing, exposed to guinea pig complement. Cytolytic "injury" was measured by the percentage of labeled cellular proteins released into the medium. Sera from sequential bleedings from eight normal Wistar rats and three rats from a nondiabetic BB subline were assayed to establish basal control cytolytic activity. The mean response +/- SD obtained with all control sera was 7.7 +/- 1.7%. A response exceeding the mean + 3 SD (12.8%) was considered significantly different from the basal value. Thirteen of the fourteen BB rats developed strongly positive sera. The cytolytic activity preceded the onset of overt diabetes. In several rats CFA appeared 4-8 wk preceding diabetes while in other rats CFA appeared 1-2 wk preceding the manifestation of the disease. These results indicate that CFA may contribute to the destruction of pancreatic islets directly or by attracting mononuclear cells.

Elevated mRNA levels of major histocompatibility complex class II genes in lymphocytes of autoimmune BB rats.

The BB rat spontaneously develops autoimmune abnormalities such as insulin-dependent diabetes mellitus and thyroiditis. The autoimmunity of the BB rat is controlled in part by genes of the major histocompatibility complex (MHC), known as the RT1 complex in the rat, and accumulating evidence suggests the involvement of MHC class II molecules. The RT1 complex specifies two types of class II molecules, which are encoded by the loci RT1.B and RT1.D. We have determined the relative steady-state mRNA levels of the class II genes RT1.B beta, RT1.D alpha, and RT1.D beta in splenic lymphocytes from individual autoimmune BB rats of various ages and from age-matched histocompatible normal Wistar-Furth (WF) rats. The relative steady-state mRNA levels of the RT1.D alpha and RT1.D beta genes, but not of the RT1.B beta gene, were elevated approximately 2.5-fold in lymphocytes of prediabetic BB rats 45-75 days old in comparison with age-matched normal WF rats and older BB rats greater than 75 days old. In the diabetic and nondiabetic BB rats greater than 75 days old, the RT1.D alpha and RT1.D beta transcripts were found at lower normal levels, similar to that of WF rats. In contrast, the RT1.B beta transcripts were found at comparable levels in lymphocytes of the BB and WF rats at all ages examined. The increased steady-state mRNA levels of the RT1.D alpha and RT1.D beta genes in the prediabetic BB rats may reflect differences in the proportion of lymphocytes expressing these genes and thus differences in splenic lymphocyte populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor I-enhanced renal expression of osteopontin after acute ischemic injury in rats.

Pretreatment of rats with insulin-like growth factor I (IGF-I) ameliorates the course of acute ischemic renal injury. Differential display PCR was used to identify genes that are expressed in kidney after induction of acute ischemic renal injury in rats pretreated with vehicle or IGF-I. One amplification product that showed enhanced expression in kidneys of rats rendered ischemic compared to kidneys of sham-operated rats was identified as osteopontin. Sequence analysis of full-length complementary DNAs revealed a single species. Renal tissue was obtained for study 12 h and 1, 2, 3, 5, 7, 14, and 28 days postinjury. Levels of whole kidney osteopontin messenger RNA (mRNA) in rats rendered ischemic 1 day previously were elevated approximately 18-fold compared to levels measured in sham-operated controls, as determined by Northern analysis. No differences were noted 12 h postinjury. Levels of osteopontin mRNA remained elevated for 14 days after ischemia, but were no longer elevated at 28 days. IGF-I pretreatment resulted in enhanced levels of osteopontin mRNA 12 h, 1 day, and 5 days postinjury. In situ hybridization demonstrated that the elevated expression of osteopontin 1 day postinjury was localized predominantly to cells in the distal tubule and medullary thick ascending limb of Henle's loop. Immunostaining showed an identical localization for elevated protein expression. Five days postinjury, osteopontin peptide and mRNA were clearly detected in regenerating proximal tubules in addition to distal tubule and medullary thick ascending limb. We propose that endogenous osteopontin serves to promote tissue regeneration and tissue remodeling within 1 day after acute ischemic injury of kidney. IGF-I enhanced expression of osteopontin at an earlier time postischemia may ameliorate the course of injury.

Antigenic diversity within a family of M proteins from group A streptococci: evidence for the role of frameshift and compensatory mutations.

The genes (emm) encoding M proteins, from isolates of group-A streptococci (GAS) serotyped as M52, M53, M80 and M nontypeable (MNT; serologically related to M53 and M80), were examined. Characterization of emm from these GAS revealed some discrepancies with serotyping, illustrating the difficulty in serotype determination when cross-reactions occur. DNA sequences corresponding to the N-terminal region of M proteins from the isolates showed considerable similarity both in the hypervariable region and the repeat regions. We propose that these serotypes form a family of closely related M types. Frameshift mutations in the hypervariable region followed by a corrective (compensatory) frameshift were observed. This may be an effective mechanism for generating antigenic diversity in the M protein.

Equivalence classes for the double-digest problem with coincident cut sites.

When restriction sites are modeled by a random process, the number of solutions to the double-digest problem (DDP) increases exponentially with the length of the DNA molecule. Cassette transformations define equivalence classes on the set of solutions to the DDP for the case of no coincident cut sites. Pevzner (1994) completely characterized the solutions to the DDP in the case of no coincident cut sites by associating solutions to DDP with alternating Eulerian paths in an edge-bicolored graph. In this paper we extend the definition of cassettes and their transformations to the general case allowing coincident cut sites. Solutions to the DDP in the general case are again characterized by associating solutions to the DDP with alternating Eulerian cycles in an extended graph.

Rapid and long-term changes to host cytotoxic T lymphocyte precursors reactive to donor antigens caused by intravenous injection of histoincompatible lymphocytes.

It has been shown previously that a single intravenous injection of mouse F1 LNC into either parent results in a rapid reduction in the ability of the recipient to generate CTL reactive against donor antigens in an in vitro MLR. The underlying mechanism appears to be the inactivation of host CTL precursors that can recognize donor lymphocytes that have entered the recirculating pool. The donor lymphocytes may be acting as functionally deleting APC, or veto cells. Here, we have injected C57BL/6 (B6) mice with (C57BL/6 x DBA/2) F1 (F1) LNC. The CTL response against donor LNC was maximally reduced by 2 days and stayed reduced for at least 6 weeks, but ultimately recovered to normal levels. The response reduction mechanism remained operative during the period when the response was reduced: Fresh FITC-labeled B6 LNC introduced into B6 recipients of an earlier injection of F1 LNC were as effectively deleted of F1-reactive CTLp as the original host B6 LNC population, the fresh FITC-labelled B6 LNC being separated from host B6 LNC by cell sorting before testing. When B6 mice injected with F1 LNC ultimately recovered their response against F1 donor antigens, reinjection of F1 LNC did not induce a new response reduction. Instead of entering the recirculating pool, the injected F1 cells were rapidly removed by a specific immune process.

Hepatocyte growth factor inhibits apoptosis after ischemic renal injury in rats.

Several studies have determined that growth factors, including hepatocyte growth factor (HGF), have a crucial role in the regenerative process of renal tubules after ischemic or toxic insult. Recent research has ascertained that as well as necrotic cell death, there is evidence of apoptosis after an acute renal injury. We attempted to determine the effect of HGF on apoptosis after ischemic renal injury in rats. We administered HGF or vehicle to 12 rats after ischemic insult and compared them with 6 sham-operated controls. Rats were killed at 48 hours, and histopathologic assessments were performed on the renal tissue. The microscale autoradiographic method was used for qualitative analysis of DNA fragmentation. This method was chosen over the widely used ethidium bromide-staining method because it increases the sensitivity of detection of apoptotic DNA. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling histopathologic staining was used to identify apoptosis in situ. Apoptotic changes were clearly shown by electron microscopy in vehicle-treated animals. Despite showing profound evidence of tubular necrosis, apoptotic changes were markedly reduced in HGF-treated animals compared with vehicle-treated animals. DNA-laddering analysis further confirmed the antiapoptotic effect of HGF. To our knowledge, this is the first in vivo illustration of the inhibitory activity of a growth factor on apoptosis in the setting of tubular necrosis. The role of apoptosis in the setting of acute renal failure has not been elucidated; thus, additional research is necessary to determine the significance of these findings.

Acute rheumatic fever in Auckland, New Zealand: spectrum of associated group A streptococci different from expected.

Annual specific rates for acute rheumatic fever (ARF) in Auckland children less than 15 years were 22/100,000 for the years 1980 to 1984. From 1984 to 1992 the rates remained relatively constant with an average of 45 (range, 30 to 70) children annually admitted with ARF to the Auckland Children's Hospital. This study examined retrospectively Group A streptococci identified from hospitalized pediatric patients during these 9 years. The total of 2410 isolates included 32 isolates from well-documented cases of ARF and an additional 6 from siblings of cases. Results of M typing indicated that streptococci associated with ARF are generally different from those described overseas and involved types which cause more skin than throat infections in the community.

Elevated blood pressure in men accompanying patients to the obstetrician's office.

Hypertension is common and leads to increased mortality among adults; yet, one-third of hypertensive adults in the United States are unaware of their condition. The purpose of this study was to determine the frequency of unrecognized elevated blood pressure (BP) in men accompanying pregnant women to the obstetrician's office. Blood pressure measurements were offered to men accompanying pregnant women to four obstetrics practices in St. Louis, Missouri. Age, race, history of hypertension, and relationship to the pregnant woman were also recorded. A total of 191 men participated in the study. Participants' ages ranged from 15 to 69 years, with a mean of 27 years. Elevated BP (> 140/90 mm Hg) was detected in 40 men (21%). Only 5% of men with an elevated BP were aware of a prior history of elevated BP. We conclude that the obstetrician's office provides a good opportunity for initial screening for hypertension in men. Follow-up is necessary to determine the accuracy of the diagnosis.

Insulin-like growth factor-I attenuates delayed graft function in a canine renal autotransplantation model.

BACKGROUND: Insulin-like growth factor-I (IGF-I) has been shown to accelerate recovery in animal models of ischemic or toxic acute renal injury. Ischemic renal injury is frequently encountered after cadaveric transplantation manifested as delayed graft function. This study was performed to determine whether perfusion of kidneys with preservation solution supplemented with IGF-I would improve the course of renal injury in a canine autotransplantation model of delayed graft function. METHODS: Dogs underwent unilateral nephrectomy with kidneys perfused and stored in Euro-Collins solution supplemented with vehicle (n = 11) or IGF-I (n = 8). After 24 hours of kidney preservation, a contralateral nephrectomy was performed and the stored kidney was autotransplanted. Renal function was examined for 5 days after the transplantation, and an inulin clearance was obtained at the time of death. RESULTS: Compared with dogs that received kidneys preserved in the vehicle, dogs receiving the IGF-I preserved kidneys had significantly lower daily serum creatinine and blood urea nitrogen levels during the course of 5 days after transplantation. Inulin clearance at death was nearly double in the IGF-I treated animals compared with the vehicle-treated controls (1.37 +/- 0.16 ml/min/kg versus 0.77 +/- 0.13 ml/min/kg; p < 0.05). CONCLUSIONS: Perfusion and storage of kidneys with preservation solution supplemented with IGF-I can attenuate the course of delayed graft function in a canine renal autotransplantation model. IGF-I may have potential for use in cadaveric human renal transplantation.

Opacity factor from group A streptococci is an apoproteinase.

Opacity factor (OF) is an enzyme, elaborated by certain serotypes of group A streptococci, which produces opalescence in mammalian sera. OF has been designated a lipoproteinase. Lipoproteins are complex structures and many enzymes are involved in their catalysis. We therefore set out to establish which of the many enzymes OF could be. Results showed that OF rendered high density lipoprotein (HDL) insoluble, accounting for the opalescence in serum, and altered its electrophoretic mobility. Electron microscopy revealed that OF caused an aggregation of HDL and an alteration in molecule shape. OF specifically split apoprotein AI of HDL into two fragments demonstrable by SDS-PAGE. We therefore designate OF as an apoproteinase.

Clonal differences within M-types of the group A Streptococcus revealed by pulsed field gel electrophoresis.

Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.