Early splenomegaly and septicaemia in homozygous sickle cell disease: A birth cohort study.

BACKGROUND: Patients with homozygous sickle cell disease (HbSS) and clinical splenomegaly by 6 months of age appeared at greater risk of invasive infections after 5 years of the Jamaican Cohort Study. We determined whether this risk remained significant over a longer study period, using a more rigorous definition of infection and examining the contribution of potential confounders. METHODS: Newborn screening of 100,000 consecutive deliveries during 1973-1981 detected 311 births with HbSS. Age at first clinical splenomegaly was used to categorize 285 of these patients in whom this could be determined: at or before 7 months (early), after 7 months (later), or 'never' palpated despite repeated examinations. Infective episodes were confined to 'first infections confirmed by positive culture'. Using a generalized linear model, the risk of septicaemia was assessed in each group, after adjusting for potential confounders. RESULTS: Of 93 'first infections', 42 occurred in 105 subjects in the 'early' group, 49 in 157 subjects in the 'later' group, and two in 23 subjects in the 'never' group; the observed to expected ratio of 1.42, 0.90 and 0.22 was highly significant (p = .003). Assessed as risk ratios, 'early' splenomegaly had a significantly higher risk ratio (RR) for septicaemia (RR = 7.4, confidence interval [CI]: 1.1-50.7, p < .05) when compared to the 'never' group adjusting for vaccine exposure and foetal haemoglobin concentration. The most common organisms were Streptococcus pneumoniae, Salmonella species, Haemophilus influenzae and Staphylococcus aureus. CONCLUSION: Early clinical splenomegaly in HbSS remains a predictor of septicaemia, defining a group that may require closer monitoring.

Conformations of peptide fragments from the FK506 binding protein: comparison with the native and urea-unfolded states.

The helix-forming tendency of seven peptide fragments corresponding with the entire sequence of the FK506 binding protein (FKBP) has been investigated in aqueous buffer and in 2,2,2-trifluoroethanol (TFE) using CD and NMR spectroscopy. All fragments exhibited random coil conformations in aqueous buffer, whereas the amount of helix induced in the peptide fragments by TFE varied. The fragment with the highest degree of helicity in TFE corresponded with the single (alpha-helix in native FKBP. Fragments corresponding with beta-strands 2 and 3 also exhibited strong propensity towards helix formation. In contrast, the fragment corresponding with beta-strand 1 did not form helix in TFE. The inherent helix-forming tendencies are interpreted in light of the native structure to suggest possible folding nucleation sites. Conformational sampling in each peptide fragment was also compared with that observed in urea-denatured FKBP. With the exception of the fragment corresponding with beta-strand 2, the formation of helical structures in the peptide fragments in TFE was correlated with the observation of turn and/or helix conformers in urea-unfolded FKBP. Surprisingly, peptide fragments in aqueous solution were less structured than the corresponding regions in urea-denatured FKBP. The conformational differences between the peptide fragments and unfolded FKBP were not due to the urea buffer or to differences in their rotational correlation times. We conclude that local amino acid interactions are not generally sufficient to account for the formation of non-random conformations in unfolded FKBP. Formation of non-random structures in unfolded FKBP may require stabilization of incipient turn or helical conformations through transient contact with non-local non-polar residues.

Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride.

Characterizing the structure properties of unfolded proteins is important for understanding the stability and folding of native proteins. However, little structural information is available for the unfolded state. Using recently developed heteronuclear multi-dimensional NMR techniques, the 1H, 13C and 15N chemical shift assignments of the FK506 binding protein (FKBP) unfolded in concentrated urea and guanidine hydrochloride (GuHCl) solutions have been obtained, and the structural properties of unfolded FKBP have been characterized. FKBP displays extensive conformational averaging when unfolded in urea and GuHCl, but defined regions of secondary structure are present. Subtle differences regarding the location and stability of the secondary structures exist between the two solvents. Secondary structure formation in unfolded FKPB was correlated with statistical and thermodynamic predictions of helix formation as well as with the three-dimensional structure of folded FKBP determined by NMR and X-ray crystallography. Residues involved in secondary structures in unfolded FKBP are generally found in the same type of secondary structure in the folded protein. An exception to this was found at the C terminus of FKBP, which forms a different secondary structure in the unfolded and folded states.

Ultra-wide bore 900 MHz high-resolution NMR at the National High Magnetic Field Laboratory.

Access to an ultra-wide bore (105 mm) 21.1 T magnet makes possible numerous advances in NMR spectroscopy and MR imaging, as well as novel applications. This magnet was developed, designed, manufactured and tested at the National High Magnetic Field Laboratory and on July 21, 2004 it was energized to 21.1 T. Commercial and unique homebuilt probes, along with a standard commercial NMR console have been installed and tested with many science applications to develop this spectrometer as a user facility. Solution NMR of membrane proteins with enhanced resolution, new pulse sequences for solid state NMR taking advantage of narrowed proton linewidths, and enhanced spatial resolution and contrast leading to improved animal imaging have been documented. In addition, it is demonstrated that spectroscopy of single site (17)O labeled macromolecules in a hydrated lipid bilayer environment can be recorded in a remarkably short period of time. (17)O spectra of aligned samples show the potential for using this data for orientational restraints and for characterizing unique details of cation binding properties to ion channels. The success of this NHMFL magnet illustrates the potential for using a similar magnet design as an outsert for high temperature superconducting insert coils to achieve an NMR magnet with a field >25 T.

N-terminal extension changes the folding mechanism of the FK506-binding protein.

Many of the protein fusion systems used to enhance the yield of recombinant proteins result in the addition of a small number of amino acid residues onto the desired protein. Here, we investigate the effect of short (three amino acid) N-terminal extensions on the equilibrium denaturation and kinetic folding and unfolding reactions of the FK506-binding protein (FKBP) and compare the results obtained with data collected on an FKBP variant lacking this extension. Isothermal equilibrium denaturation experiments demonstrated that the N-terminal extension had a slight destabilizing effect. NMR investigations showed that the N-terminal extension slightly perturbed the protein structure near the site of the extension, with lesser effects being propagated into the single alpha-helix of FKBP. These structural perturbations probably account for the differential stability. In contrast to the relatively minor equilibrium effects, the N-terminal extension generated a kinetic-folding intermediate that is not observed in the shorter construct. Kinetic experiments performed on a construct with a different amino acid sequence in the extension showed that the length and the sequence of the extension both contribute to the observed equilibrium and kinetic effects. These results point to an important role for the N terminus in the folding of FKBP and suggest that a biological consequence of N-terminal methionine removal observed in many eukaryotic and prokaryotic proteins is to increase the folding efficiency of the polypeptide chain.

Structure and stability effects of mutations designed to increase the primary sequence symmetry within the core region of a beta-trefoil.

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil hyperfamily and exhibits a characteristic threefold symmetry of the tertiary structure. However, evidence of this symmetry is not readily apparent at the level of the primary sequence. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary sequence during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to threefold symmetry at the level of the primary sequence. The consequences of these mutations regarding structure and stability were evaluated using a combination of X-ray crystallography and differential scanning calorimetry. The mutational effects on structure and stability can be rationalized through the characterization of "microcavities" within the core detected using a 1.0A probe radius. The results show that the symmetric constraint within the primary sequence is compatible with a well-packed core and near wild-type stability. However, despite the general maintenance of overall thermal stability, a noticeable increase in non-two-state denaturation follows the increase in primary sequence symmetry. Therefore, properties of folding, rather than stability, may contribute to the selective pressure for asymmetric primary core sequences within symmetric protein architectures.

Human recombinant [C22A] FK506-binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR.

Hydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentation, combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry (MS). After a specified period of H/D exchange in solution, C22A FKBP was digested by pepsin under slow exchange conditions (pH 2.4, 0 degree C), and then subjected to on-line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independently by heteronuclear two-dimensional NMR on 15N-enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive the distributions of hydrogen exchange rate constants from the MS-determined deuterium exchange-in curves in either the holoprotein or its proteolytic segments. The MEM-derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate constants determined by NMR for individual amide protons. The rate constant distributions determined by both methods are consistent and complementary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins.

Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments.

Two multi-dimensional heteronuclear NMR experiments are described for assigning the resonances in uniformly 15N- and 13C-labeled proteins. In one experiment (HCNH-TOCSY), the amide nitrogen and proton are correlated to the side-chain protons and carbons of the same and preceding residue. In a second triple resonance experiment (HC(CO)NH-TOCSY), the amide nitrogen and proton of one residue is correlated exclusively with the side-chain proton and carbon resonances of the preceding residue by transferring magnetization through the intervening carbonyl. The utility of these two experiments for making sequential resonance assignments in proteins is illustrated for [U-15N,13C]FKBP (107 residues) complexed to the immunosuppressant, ascomycin.

Experimental infection of six species of ixodid ticks with Dugbe virus (family Bunyaviridae, genus Nairovirus).

The vector potential of each of 6 species of colonized North American and African ixodid ticks was assessed by intracoelomic inoculation with Dugbe virus (IbAr 1792, 14th passage in suckling mouse brain) and viral titers were monitored after selected incubation periods. Persistence of Dugbe virus for greater than or equal to 53 days in 5 species (Dermacentor andersoni, D. variabilis, Amblyomma americanum, Rhipicephalus appendiculatus, and R. sanguineus) indicates that infection occurred. Viral titers were significantly higher in female vs. male D. variabilis, R. appendiculatus, and A. americanum after blood feeding. Blood feeding had no significant effect on the viral titers of either female or male R. sanguineus. D. andersoni males also exhibited no significant change in viral titers after blood-feeding, but 100% (20/20) of drop-off females and 96% (24/25) of post-oviposition females (36 days postinoculation) contained no detectable virus even though virus was still found in unfed specimens less than or equal to 124 days postinoculation. Virus was not recovered from greater than 30,000 1st generation progeny (eggs, larvae, nymphs, adults) collected as eggs from inoculated female D. andersoni, D. variabilis, R. sanguineus, and R. appendiculatus 27-51 days postinoculation. R. sanguineus and R. appendiculatus transmitted Dugbe virus to guinea pigs when allowed to feed 1-3 weeks postinoculation.

Experimental transmission of Crimean-Congo hemorrhagic fever virus by Hyalomma truncatum Koch.

Larval Hyalomma truncatum ticks were infected with Crimean-Congo hemorrhagic fever (CCHF) virus by allowing them to engorge on viremic newborn mice. The overall tick infection rate was 4.4% (24/542). Virus was detected in specimens for greater than or equal to 160 days postinfection. Transstadial transmission to the adult tick stage was observed and horizontal transmission to a mammalian host was demonstrated. Horizontal transmission of CCHF virus to uninfected adult ticks occurred while feeding with transstadially infected ticks on the same host. No evidence of transovarial virus transmission from infected female ticks to their 1st generation progeny was observed.

Transstadial and horizontal transmission of Rift Valley fever virus in Hyalomma truncatum.

We exposed Hyalomma truncatum and Rhipicephalus appendiculatus to Rift Valley fever (RVF) virus in order to assess the possible role of these ticks as enzootic/epizootic RVF vectors. The virus replicated in H. truncatum after intracoelomic inoculation, and a minimum transmission rate of 17% was achieved after 15 days intrinsic incubation. The virus persisted at least 58 days in these ticks. Virus was also shown to pass transstadially from inoculated H. truncatum nymphs to adults, with peak viral titers reaching 10(3.5) plaque-forming units (PFU) in adult males after they were provided with bloodmeals. Virus was recovered from adult females 121 days after they were inoculated as nymphs. Viral titers peaked in inoculated male ticks after dropping off a host (mean titer = 10(4.3) PFU). RVF virus was not detected in pools of eggs and larval progeny from 11 infected female H. truncatum. H. truncatum larvae and nymphs did not become infected after ingesting greater than 10(2.0) PFU while feeding on a RVF viremic hamster. The number of infected specimens declined rapidly after RVF virus was inoculated into R. appendiculatus adults, and virus was undetectable 12 days post-inoculation.

Antigen-capture enzyme-linked immunosorbent assay for detection and quantification of Crimean-Congo hemorrhagic fever virus in the tick, Hyalomma truncatum.

A viral antigen-capture ELISA was compared to a viral plaque-assay on human cell monolayers for detection and quantification of Crimean-Congo hemorrhagic fever virus in triturated experimentally infected Hyalomma truncatum ticks. In suspensions of ticks exposed as larvae to viremic mice, the ELISA detected 13% positive as compared to 3% (n = 721) positive by plaque-assay. Adult ticks inoculated with virus and sampled up to 102 days later were 84% positive by ELISA compared to 36% (n = 273) positive by plaque-assay. The two tests detected similar proportions of positive ticks in the two weeks immediately after viral inoculation; however, the ELISA was positive in 100% of inoculated adult ticks from 18-102 days post-inoculation while the plaque-assay was positive in 33% (n = 135) of the same specimens. CCHF viral antigen was detected in 10% (n = 101) of first-generation progeny tested as unfed larva pools by ELISA, yet no virus was detected by plaque-assay, indicating that either non-infective viral fragments or very low levels of live virus were detected by ELISA in these tick progeny. As detected by plaque assay, virus inoculated unfed adult ticks were virtually all infected by day 8 post-inoculation; by day 21 post-inoculation only 33% were detected as positive. A cohort of these ticks were allowed to blood feed from day 21-31 post-inoculation. When assayed after feeding all female ticks and nearly 50% of male ticks were detected as virus-positive. This indicates that the virus likely persisted in the unfed ticks below the level of detectability of the plaque-assay and increased in the blood fed ticks up to a detectable level. The ELISA however, detected 100% of ticks as virus-positive from day 14 post-inoculation throughout the remainder of the study, regardless of feeding status (day 102 post-inoculation). These results indicate that antigen-detection ELISA is more sensitive in detecting CCHF virus in ticks than plaque-assay. Since an infected tick remains antigen-positive by ELISA for possibly the remainder of its life, this assay will be a major improvement in field surveys and vector competency studies of ticks for CCHF virus.

Feeding nymphs of Hyalomma truncatum (Acari: Ixodidae) on guinea pigs previously exposed to nymphs.

The ability of nymphal Hyalomma truncatum Koch to feed on guinea pigs previously exposed to nymphal ticks was studied by measuring the percentage of ticks engorging and molting, their engorged weight, and hemoglobin content. Four guinea pigs were infested with 20, 40, 80, and 200 nymphs, respectively, on three occasions at 21-d intervals, followed by a fourth infestation after another 21-d interval with 100 nymphs. Guinea pigs originally infested with 20 and 80 nymphs were infested a fifth time with 100 nymphs 3 mo after the fourth infestation. Guinea pigs originally infested with 40 and 200 nymphs were infested a fifth time with 100 nymphs 6 mo after the fourth infestation. There was a significant decrease, below infestation I levels, in the number of ticks engorging on each guinea pig during infestations IV and V. Whereas all nymphs molted after infestation I, the percentage of nymphs molting after infestation V ranged from 0 to 34%. For ticks infested on each of the guinea pigs, the percentage molting after infestations III-V were significantly lower than those observed after infestation I. A significant decrease in the mean weight of engorged ticks was observed starting at infestation III. Mean hemoglobin content of ticks declined significantly at infestation II. The response elicited by feeding nymphs appeared to last at least 6 mo.

Weight gain, hemoglobin uptake, and virus ingestion by Hyalomma truncatum (Acari: Ixodidae) ticks after engorgement on viremic guinea pigs.

The ability of guinea pigs to serve as a source of an arbovirus for feeding ticks was examined with Hyalomma truncatum Koch and Venezuelan equine encephalomyelitis virus. Ticks fed on guinea pigs to varying degrees, as indicated by both low and high weight gain and hemoglobin uptake. Pools of larval ticks measured at drop-off with undetectable or very low hemoglobin levels (mean hemoglobin content = 0.05 mg per pool) contained the same amount of virus (> 10(3.0) plaque-forming units [PFU] per pool) as pools of ticks with high hemoglobin levels (mean hemoglobin = 0.15 mg per pool). A group of nymphs that ingested a mean of 0.13 mg of hemoglobin contained the same amount of virus (mean viral titer approximately 10(2.5) PFU) as a group of nymphs that ingested 0.32 mg of hemoglobin. Some adult ticks that ingested < 0.35 mg of hemoglobin contained more virus than adults that ingested > 4.7 mg of hemoglobin.

Association of ticks (Acari: Ixodoidea) with rodent burrows in northern Senegal.

Four species of ticks were retrieved from burrows of 64 multimammate rats, Mastomys erythroleucus (Temminck), 55-gerbils, Taterillus gracilis (Thomas) or T. pygargus (Cuvier), 13 Nile rats, Arvicanthis niloticus (DeMarest), and five Geoffroy's ground squirrels, Xerus erythropus (Geoffroy) from May 1987 through August 1988 at two study sites in northern Senegal. Ornithodoros sonrai Sautet & Witkowski were recovered from 95% (74/78) of burrows near Bandia and 6% (4/66) of burrows near Yonofere. Eight Hyalomma truncatum Koch, four Rhipicephalus guilhoni Morel & Vassiliades, and one R. sulcatus Neumann were recovered from 144 rodent burrows (nine tick-positive burrows) from both locations. No seasonal trend of tick abundance or activity was noted, nor was any pattern of burrow preference by ticks detected. Rodent burrows in either location appeared to be little used by ixodid ticks.

Feeding efficiency of larval Hyalomma truncatum (Acari: Ixodidae) on hosts previously exposed to ticks.

The effect of guinea pig response to feeding larval Hyalomma truncatum Koch ticks was studied by measuring the percentage of ticks engorging and molting, their engorged weight, and hemoglobin content. Four guinea pigs were infested with 100, 200, 400, and 1,000 larvae, respectively, on three occasions at 21-d intervals, followed by a fourth infestation with 500 larvae. During the second, third, and fourth infestations, significantly fewer ticks engorged on each guinea pig than during the first infestation. The greatest reduction in percentage molting occurred during the fourth infestation on the animal with successive exposure to 400 larvae; only 24% of the ticks that fed molted. Ticks with the lowest mean weight and hemoglobin content also engorged on this animal during the fourth infestation. Guinea pigs exposed to 200 and 400 H. truncatum larvae elicited the greatest change in feeding efficiency during the fourth infestation. However, these hosts had no effect on a single subsequent fifth infestation with Amblyomma cajennense (F.) nymphs, as greater than 95% successfully engorged.

Experimental vector incompetence of a soft tick, Ornithodoros sonrai (Acari: Argasidae), for Crimean-Congo hemorrhagic fever virus.

Adults and nymphs of a soft tick, Ornithodoros sonrai Sautet & Witkowski, were allowed to feed on suckling mice that had been experimentally infected with Crimean-Congo hemorrhagic fever (CCHF) virus (IbAr 10200 strain). The mean viral titer of mouse blood at the time of tick feeding was 10(3.2) plaque-forming units (PFU) per ml. Samples of ticks were assayed on 12 occasions between days 0 and 31 after the viremic blood meal. Mean CCHF viral titers were 10(2.1) PFU per tick immediately after the viremic meal but declined to 10(1.2) PFU per tick after 2 d, and no virus was detected beyond 8 d. The percentage of ticks with detectable virus was 92% (22/24) immediately after the viremic meal, but then declined to 20% (2/10) after 4 d and to 0% (0/44) after 11 or more days. Ticks were allowed to feed on sets of three naive suckling mice on days 0, 2, 5, 8, 11, 14, 21, and 28 after the viremic blood meal, but CCHF viral transmission did not occur. Similarly, no transovarial transmission of virus from CCHF virus-exposed O. sonrai to their progeny was observed. These results strongly indicate that O. sonrai is not a vector of CCHF virus.

Laboratory biology of Hyalomma truncatum (Acari: Ixodidae).

The life cycle of Hyalomma truncatum Koch (Acari: Ixodidae) required an average of 108 d at 26 +/- 1 degree C. 92-96% RH, and a 12:12 (L:D) photoperiod to complete. Mean weights of unfed larvae, nymphs, and females were 0.02, 0.19, and 11.1 mg, respectively. Weight of larvae, nymphs, and females increased 20-, 91-, and 48-fold, respectively, as a result of feeding on guinea pigs. All stages exhibited host-seeking behavior less than 1 d after emergence. The mean (+/- SE) feeding period of larvae, nymphs, and adults was 3.8 (+/- 0.1), 7.7 (+/- 0.3), and 8.3 (+/- 0.3) d, respectively. Larvae and nymphs molted an average of 11.0 (+/- 0.3), and 30.7 (+/- 0.2) d after engorgement, respectively. The female/male sex ratio, as determined from emerged adults, was 1.4:1. Oviposition started an average of 11.9 (+/- 0.8) d after engorgement, and a mean of 6.701 eggs per female was deposited. A total of only 48% of the eggs enclosed after a mean incubation of 35 (+/- 1.1) d. Females converted 56% of their engorged weight into eggs and produced an average of 12,614 (+/- 2.0) eggs/g of engorged body weight. On the first day of oviposition, an egg less than 24 h old weighed an average of 44.8 (+/- 1.5) micrograms. Egg weight was significantly (P less than 0.01) lower during peak egg production (days 2-8 after onset of oviposition) than during reduced egg production (days 14-20 after onset of oviposition).

Laboratory transmission of Sindbis virus by Aedes albopictus, Ae. aegypti, and Culex pipiens (Diptera: Culicidae).

The susceptibility of Aedes albopictus (Skuse) for Sindbis (SIN) virus was examined in the laboratory. Ae. albopictus, Ae. aegypti (L.), and Culex pipiens (L.) became infected with and subsequently transmitted SIN virus by bite to chicks after feeding on viremic 1-d-old chicks. After ingesting 10(5.3) plaque-forming units (PFU)/ml, Ae. albopictus had the highest transmission rate (30%) of the 3 species. Transmission by Ae. aegypti was less efficient (7%), whereas none of the Cx. pipiens transmitted virus. Transmission rates were higher for Ae. albopictus (53%) and Cx. pipiens (37%) when they fed on chicks with a viremia of 10(8.4) PFU/ml. Ae. aegypti was not tested at this dose. Based on these studies, the increased geographic distribution of Ae. albopictus, and its opportunistic feeding behavior, this species should be considered as a potential bridge vector of SIN virus.

Transmission of Crimean-Congo hemorrhagic fever virus by Hyalomma impeltatum (Acari:Ixodidae) after experimental infection.

We examined the potential for Hyalomma impeltatum Schulze & Schlottke ticks to transmit Crimean-Congo hemorrhagic fever (CCHF) virus. After feeding on 1-d-old mice that had been inoculated with CCHF virus, larval H. impeltatum became infected with and subsequently transmitted CCHF virus transstadially to nymphs. These nymphs transmitted virus horizontally to guinea pigs and transstadially to adults. A minimum of 13% of the adult H. impeltatum, exposed to virus as larvae, transmitted virus by bite to guinea pigs. An enzyme-linked immunosorbent assay detected CCHF viral antigen in 63% of the adult ticks derived from those exposed to this virus as larvae. This tick species should be considered as a potential vector of CCHF virus.