Cord blood Streptococcus pneumoniae-specific cellular immune responses predict early pneumococcal carriage in high-risk infants in Papua New Guinea.

In areas where Streptococcus pneumoniae is highly endemic, infants experience very early pneumococcal colonization of the upper respiratory tract, with carriage often persisting into adulthood. We aimed to explore whether newborns in high-risk areas have pre-existing pneumococcal-specific cellular immune responses that may affect early pneumococcal acquisition. Cord blood mononuclear cells (CBMC) of 84 Papua New Guinean (PNG; high endemic) and 33 Australian (AUS; low endemic) newborns were stimulated in vitro with detoxified pneumolysin (dPly) or pneumococcal surface protein A (PspA; families 1 and 2) and compared for cytokine responses. Within the PNG cohort, associations between CBMC dPly and PspA-induced responses and pneumococcal colonization within the first month of life were studied. Significantly higher PspA-specific interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-5, IL-6, IL-10 and IL-13 responses, and lower dPly-IL-6 responses were produced in CBMC cultures of PNG compared to AUS newborns. Higher CBMC PspA-IL-5 and PspA-IL-13 responses correlated with a higher proportion of cord CD4 T cells, and higher dPly-IL-6 responses with a higher frequency of cord antigen-presenting cells. In the PNG cohort, higher PspA-specific IL-5 and IL-6 CBMC responses were associated independently and significantly with increased risk of earlier pneumococcal colonization, while a significant protective effect was found for higher PspA-IL-10 CBMC responses. Pneumococcus-specific cellular immune responses differ between children born in pneumococcal high versus low endemic settings, which may contribute to the higher risk of infants in high endemic settings for early pneumococcal colonization, and hence disease.

Effects of Bt cotton and crylac toxin on survival and development of pink bollworm (Lepidoptera: Gelechiidae).

We evaluated the effects of Bacillus thuringiensis (Bt) toxin CrylAc on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinophora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in CrylAc concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces CrylAc (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.

Overwintering cost associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae).

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent.

Fetal death. A 10-year retrospective study.

Fetal death has been defined by the World Health Organization as death before complete expulsion or extraction from its mother of a product of conception, irrespective of the duration of pregnancy. Certain causes of fetal death, including syphilis, Rh isoimmunization, toxemia, and diabetes, have shown significant declines over the past several decades. However, many fetal losses continue to occur from intrauterine infections, lethal malformations, fetal growth retardation, and abruptio placentae. Fetal death with no identifiable specific cause is another consideration when dealing with these cases. Other risk factors can include maternal, sociodemographic, and medical care factors. The authors reviewed all forensic cases referred for autopsy to the Forensic Section of the Medical University of South Carolina, Medical Examiners' Office over the 10-year period 1990-1999. All cases listed as fetal death or stillbirth were included. The 42 cases were analyzed as to fetus' gestational age, sex, race, weight, location of delivery, history of prenatal care, maternal drug use, chromosomal abnormalities, cause and manner of death, and autopsy findings. The black:white ratio was approximately 2:1, and the male:female ratio was virtually 1:1. Most fetuses were older than 20 weeks' gestational age, with one third between 20 and 29 weeks. The majority were externally normal aside from maceration. Only 7.5% had congenital anomalies. Twenty-one of 38 placentas were grossly and microscopically normal. Of cases with toxicologic analysis, 21% were positive for drugs, and 17% were positive for cocaine/benzoylecgonine. The manner of death was classified as natural (28), accident (2), and undetermined (12). Few studies have reported the specific causes of fetal death, and the lack of uniformity in data collection and classification of causes of fetal death has made comparisons difficult. The authors present this retrospective study to better determine the factors leading to fetal demise in the hope of assisting death investigators in this challenging arena.

Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae).

Transgenic cotton producing a Bacillus thuringiensis (Bt) toxin is widely used for controlling the pink bollworm, Perctinophora gossypiella (Saunders). We compared performance of pink bollworm strains resistant to Bt cotton with performance of their susceptible counterparts on non-Bt cotton. We found fitness costs that reduced survival on non-Bt cotton by an average of 51.5% in two resistant strains relative to the susceptible strains. The survival cost was recessive in one set of crosses between a resistant strain and the susceptible strain from which it was derived. However, crosses involving an unrelated resistant and susceptible strain indicated that the survival cost could be dominant. Development time on non-Bt cotton did not differ between the two related resistant and susceptible strains. A slight recessive cost affecting development time was suggested by comparison of the unrelated resistant and susceptible strains. Maternal effects transmitted by parents that had eaten Bt-treated artificial diet as larvae had negative effects on embryogenesis, adult fertility, or both, and reduced the ability of neonates to enter cotton bolls. These results provide further evidence that fitness costs associated with the evolution of resistance to Bt cotton are substantial in the pink bollworm.

Frequency of resistance to Bacillus thuringiensis in field populations of pink bollworm.

Strategies for delaying pest resistance to genetically modified crops that produce Bacillus thuringiensis (Bt) toxins are based primarily on theoretical models. One key assumption of such models is that genes conferring resistance are rare. Previous estimates for lepidopteran pests targeted by Bt crops seem to meet this assumption. We report here that the estimated frequency of a recessive allele conferring resistance to Bt toxin Cry1Ac was 0.16 (95% confidence interval = 0.05-0.26) in strains of pink bollworm (Pectinophora gossypiella) derived from 10 Arizona cotton fields during 1997. Unexpectedly, the estimated resistance allele frequency did not increase from 1997 to 1999 and Bt cotton remained extremely effective against pink bollworm. These results demonstrate that the assumptions and predictions of resistance management models must be reexamined.

Cloning and characterisation of ITGAV, the genomic sequence for human cell adhesion protein (vitronectin) receptor alpha subunit, CD51.

The integrin family of receptors serves as major receptors for extracellular matrix-mediated cell adhesion and migration, cytoskeletal organisation, cell proliferation, survival, and differentiation. The alpha-V integrins consist of a subset which share a common alpha-V subunit combined with one of five beta subunits (beta-1, 3, 5, 6, or 8). The alpha-V integrins have been implicated in a number of developmental processes, including vasculogenesis and angiogenesis, and are therapeutic targets for inhibition of angiogenesis and osteoporosis. The human cDNA for alpha-V integrin (ITGAV) consists of a 5,717-bp transcript with a coding sequence (CDS) of 3,146 bp encoding a 150-kDa mature peptide. Here we describe the gene structure of ITGAV.

Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice.

Application of USAF G-suit technology for clinical orthostatic hypotension: a case study.

INTRODUCTION: The purpose of this study was to determine the effectiveness of a USAF anti-gravity suit (G-suit) on the stability of a patient with chronic orthostatic hypotension. METHODS: A 37-yr-old female with a history of insulin-dependent diabetes mellitus (IDDM) and symptomatic orthostasis was evaluated and the results were compared with those of non-diabetic controls, matched for age, height, and weight. Cardiac vagal tone was assessed by determination of standard deviation of 100 R-R intervals (R-R SD). We assessed the carotid-cardiac baroreflex response by plotting R-R intervals (ms) at each of eight neck pressure steps with their respective carotid distending pressures (mm Hg). Heart rate and blood pressure were recorded in response to the Valsalva maneuver (VM) performed at an expiratory pressure of 30 mmHg to assess integrated baroreflex responses. Blood pressures and heart rate were measured during three 5-min stand tests to assess orthostatic responses: a) without G-suit; b) with noninflated G-suit; and c) with inflated G-suit (50 mm Hg). RESULTS: The IDDM patient had minimal baseline cardiac vagal tone (R-R SD = 8.5 ms) compared with the average response of a control group of 24 subjects with orthostatic stability (R-R SD = 67.2 +/- 7.1 ms). Carotid-cardiac baroreflex response was virtually non-existent in the IDDM patient (Gain = 0.06 ms.mm Hg-1) compared to the control subjects (4.4 +/- 0.8 ms.mm Hg-1). VM responses corroborated the lack of cardiac baroreflex response in the IDDM patient, while blood pressure changes during the VM were similar to those of the controls. Upon standing, the IDDM patient demonstrated severe orthostatic hypotension (90 mm Hg SBP) and tachycardia without the G-suit. The G-suit, with and without pressure, reduced hypotension and tachycardia during standing. CONCLUSION: These results demonstrate successful application of Air Force technology as a useful alternative to pharmacologic intervention in the treatment of a patient with autonomic dysfunction leading to supine hypertension and orthostatic hypotension.

Hyperglycemia inhibits mechanoreceptor-mediated gastrocolonic responses and colonic peristaltic reflexes in healthy humans.

BACKGROUND/AIMS: The effects of hyperglycemia on colonic motor function are unknown. Therefore, colonic neuromuscular function was tested in normal volunteers as a model for constipation in diabetes. METHODS: Extended (gastrocolonic response) and local (peristaltic reflex) neural responses and colonic muscle contractility were tested under control, hyperglycemic clamp, and euglycemic, hyperinsulinemic clamp conditions with placement of barostat-regulated balloons in the descending colon to measure changes in tone as differences in balloon volume. RESULTS: Hyperglycemic clamping to 274 +/- 3 mg/dL blunted increases in colon tone evoked by gastric distention (gastrocolonic response) (100-300 mL) but did not affect gastric tone. Three descending colonic balloons in series assessed the peristaltic reflex. Inflation of the middle stimulus balloon increased proximal tone, an increase that was blunted by hyperglycemia, but produced distal relaxation followed by increases in tone that were unaffected by hyperglycemia. Euglycemic, hyperinsulinemic clamping had no effect on the gastrocolonic response or peristaltic reflex. Tonic increases evoked by bethanechol (5 mg administered subcutaneously) were unaffected by hyperglycemic clamping. CONCLUSIONS: Hyperglycemia blunts mechanoreceptor-mediated gastrocolonic responses and ascending contractions but not descending components of the peristaltic reflex in humans, effects not caused by hyperinsulinemia or direct muscle actions. These inhibitory effects on long and short neural reflexes that modulate colonic motility may contribute to constipation in diabetes.

Parallel temperature dependence of contracture-associated enzyme release due to anoxia, 2,4-dinitrophenol (DNP), or caffeine and the calcium paradox.

Hypothermia during calcium-free perfusion of hearts protects them from injury caused by subsequent calcium repletion at 37 C (calcium paradox). Injury to calcium-free hearts is also associated with contracture caused by anoxia, 2,4-dinitrophenol (DNP), or caffeine. This study was done for the purpose of determining whether hypothermia during calcium-free perfusions protects hearts from contracture-associated injury. Langendorff-perfused rat hearts were studied in four experimental groups: I) Anoxia: Thirty minutes of anoxic perfusion at 37 C was followed by thirty minutes of anoxic calcium-free perfusion at 37-18 C. II) Calcium paradox: Five minutes of calcium-free perfusion at 37-18 C was followed by calcium repletion at 37 C. III, IVa) Caffeine or DNP: Five minutes of calcium-free perfusion at 37-18 C was followed by addition of 10 mM caffeine or 1 mM DNP in calcium-free medium at 37 C or, IVb) 1 mM DNP in calcium-free medium at 22 C. Injury was assessed by measurement of serial releases of creatine kinase (CK) in effluents and by cellular morphology. The results show that progressive hypothermia to 22 C during calcium-free perfusion periods produced a progressive reduction of CK release and morphologic evidence of injury due to anoxia, caffeine, or DNP, which closely paralleled protection of hearts from the calcium paradox. Protection from injury in all experimental groups was associated with preservation of sarcolemmal membrane integrity and prevention of cell separations at intercalated disk junctions. It is proposed that weakening of intercalated disks occurs during calcium-free perfusions and may be a cause of mechanical fragility of the sarcolemma. Hypothermia may protect hearts from contracture-associated injury by preserving the integrity of intercalated disk junctions during periods of extracellular calcium depletion.

Mechanism of enzyme release in the calcium paradox.

Calcium-free rat hearts develop separations of fascia adherens junctions of intercalated discs. Such hearts are susceptible to membrane injury and enzyme release during anoxic contracture. Anoxic enzyme release was exacerbated by distention of heart ventricles with a balloon. Dinitrophenol (DNP) and caffeine were used to induce contracture in calcium-free hearts. Both DNP and caffeine caused a massive enzyme release from calcium-free but not from control hearts. Caffeine-induced enzyme release occurred despite Amytal inhibition of mitochondrial respiration. These results demonstrate that in calcium-free hearts with contracture or ventricular distention, enzyme release occurred without calcium repletion, from hearts depleted of ATP and in the absence of mitochondrial function. A relationship between contracture-mediated enzyme release and the calcium paradox was suggested by studies of the effects of hypothermia on enzyme release. Hypothermia to 22 degrees C protects hearts against both the calcium paradox and anoxic, DNP and caffeine injury. The parallel temperature dependence of protection between contracture-mediated enzyme release and the calcium paradox is evidence that contracture may also be a mediator of sarcolemmal membrane injury and enzyme release in the calcium paradox.

Physical stress-mediated enzyme release from calcium-deficient hearts.

The effects of physical stresses produced by transient distension of a left ventricular balloon, on myocardial creatine kinase (CK) release and cellular morphology, were studied in Langendorff perfused rat hearts at 37 degrees C. Hearts subjected to 15 or 30 min anoxia, developed anoxic contracture, but only small amounts of CK were released following ventricular distension. In contrast, when anoxic hearts in contracture were perfused with calcium-free medium for 5 min, prior to distension, there occurred a large peak of CK release immediately following inflation of the intraventricular balloon. Oxygenated hearts or hearts made anoxic, but which had not developed contracture, release little CK activity, although they were subjected to ventricular distension after a calcium-free perfusion period. Large myocardial enzyme releases were associated with morphological lesions of widely separated cells, dehiscence of intercalated disc cell junctions, and sarcolemmal membrane damage. Hearts subjected to calcium-free perfusion were abnormally susceptible to physical stress-induced myocardial enzyme release.

DNA methylation patterns in the 5S DNAs of Xenopus laevis.

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.

Capillary lengths and areas, and intercapillary distances in tissue near the human knee.

Quantiative stereological electron microscopy has been used to investigate the capillary lengths, surface areas and intercapillary distances in the tissues around the human knee, the synovial membrane, synovial capsule, fat and tendon. The vascularity of these regions was much less than in other areas of the body, especially muscle.