Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Dose‐intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34⁺ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum‐free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800‐fold expansion in cell number was achieved for UCB, and up to 4,000‐fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10‐fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave‐type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832–840 © 2009 Wiley Periodicals, Inc.     

Do Girls Have a Nutritional Disadvantage Compared with Boys? Statistical Models of Breastfeeding and Food Consumption Inequalities among Indian Siblings

Background

India is the only nation where girls have greater risks of under-5 mortality than boys. We test whether female disadvantage in breastfeeding and food allocation accounts for gender disparities in mortality.

Methods and Findings

Secondary, publicly available anonymized and de-identified data were used; no ethics committee review was required. Multivariate regression and Cox models were performed using Round 3 of India’s National Family and Health Survey (2005–2006; response rate = 93.5%). Models were disaggregated by birth order and sibling gender, and adjusted for maternal age, education, and fixed effects, urban residence, household deprivation, and other sociodemographics. Mothers’ reported practices of WHO/UNICEF recommendations for breastfeeding initiation, exclusivity, and total duration (ages 0–59 months), children’s consumption of 24 food items (6–59 months), and child survival (0–59 months) were examined for first- and secondborns (n = 20,395). Girls were breastfed on average for 0.45 months less than boys (95% CI: = 0.15 months to 0.75 months, p = 0.004). There were no gender differences in breastfeeding initiation (OR = 1.04, 95% CI: 0.97 to 1.12) or exclusivity (OR = 1.06, 95% CI: 0.99 to 1.14). Differences in breastfeeding cessation emerged between 12 and 36 months in secondborn females. Compared with boys, girls had lower consumption of fresh milk by 14% (95% CI: 79% to 94%, p = 0.001) and breast milk by 21% (95% CI: 70% to 90%, p<0.000). Each additional month of breastfeeding was associated with a 24% lower risk of mortality (OR = 0.76, 95% CI: 0.73 to 0.79, p<0.000). Girls’ shorter breastfeeding duration accounted for an 11% increased probability of dying before age 5, accounting for about 50% of their survival disadvantage compared with other low-income countries.

Conclusions

Indian girls are breastfed for shorter periods than boys and consume less milk. Future research should investigate the role of additional factors driving India’s female survival disadvantage.     

Infant distress vocalizations in the Southern African Lesser Bushbaby (Galago moholi)

Infant lesser bushbabies are typically able, from the earliest age, to utter distress vocalizations which appear to have major adaptive significance. Distress vocalizations of infant G. moholi were analyzed by means of a digital storage oscilloscope. Three kinds of sound analyses were carried out: analysis of the sound wave signal, sequential spectrum analysis, and fast Fourier transformation. We emphasize analysis of the frequency spectrum. Three distress calls were identified-the click call, the crackle, and the chirp-which we report for the first time. Click calls were typically composed of short, high-pitched pulses (clicks) uttered in fast, repetitive sequences with a broad band frequency spectrum containing frequencies up to 60 kHz. These acoustic characteristics appear to be particularly well suited for accurate spatial localization of the sound source. The main function of the click call is apparently to alert and to guide the mother to a lost or endangered infant. These basic acoustic characteristics-related to sound localization-are shared to some extent, with the other two distress calls. However, the three calls appear to represent different degrees of distress, with the click call being the basic, call, the crackle corresponding to a greater degree of distress, and the chirp possibly signaling extreme distress such as physical pain.We prefer the specific name,Galago moholi (A. Smith, 1836), for the southern African lesser bushbaby [instead ofGalago senegalensis moholi (A. Smith, 1839)] according to the new classification proposed by Olson (1979, 1986) and Nashet al. (1989).     

Acquisition of manganous ions by mutans group streptococci.

The cariogenic bacteria Streptococcus sobrinus and S. cricetus were shown to have an absolute requirement for manganous ion in order to bind glucans or to adhere to glass in the presence of sucrose. The bacteria possessed a reasonably high affinity transport system for 54Mn2+, yielding a Km of about 12 microM. The Vmax for uptake of 54Mn2+ in S. sobrinus was increased when the bacteria were grown in Mn-depleted medium, but the Km remained the same. There was no evidence for two Mn2+ uptake systems, commonly observed for many bacteria. Ions such as Ca2+, Co2+, Co3+, Cu2+, Fe2+, Fe3+, Hg2+, Mg2+, Ni2+, and Zn2+ did not inhibit the uptake of 54Mn2+ by the bacteria, although Cd2+ was a potent inhibitor. Fractionation experiments showed that manganese was distributed in protoplasts (67%) and in the cell wall (33%). Approximately 80% of the 54Mn2+ in S. sobrinus was rapidly exchangeable with nonradioactive Mn2+. Electron spin resonance experiments showed that all of the manganese was bound or restricted in mobility. Proton motive force-dissipating agents increased the acquisition of 54Mn2+ by the streptococci, probably because the wall became more negatively charged when the cell could no longer produce protons.     

Endogenous clock gene expression in the suprachiasmatic nuclei of previsual newborn rabbits is entrained by nursing

The rabbit is particularly suitable for investigating the development of mammalian circadian function. Blind at birth, the pups are only visited by the mother to be nursed once every 24 h for about 3 min and so can be studied largely without maternal interference. They anticipate the mother's visit with increased behavioral arousal and with a rise in body temperature, both of which represent endogenous circadian rhythms. We now report that in newborn pups the suprachiasmatic nuclei of the hypothalamus (SCN; the main circadian pacemaker in mammals) show endogenous 24‐h rhythmicity in the expression of the clock genes Per1, Per2, and Bmal1. Pups nursed from postnatal days 1 to 7 and fasted to day 9 showed the same rhythms of clock gene expression as normally nursed controls. We also report that these rhythms are entrained by nursing. Pups killed on postnatal days 3–4 showed the same rhythms in gene expression as pups in the previous experiment, whereas littermates subsequently nursed from postnatal days 4 to 7 with nursing delayed 6 h showed a corresponding shift in the diurnal pattern of clock gene expression. Consistent with this, two groups of pups implanted with telemetric thermal sensors and nursed 6 h apart had daily patterns in body temperature synchronized with the two different nursing times. We conclude that the expression of clock genes associated with the newborn rabbit's circadian system is entrained by nonphotic cues accompanying nursing, the exact nature of which now needs to be clarified. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Comparison of genetic and cytogenetic maps of hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR and DArT markers

A number of technologies are available to increase the abundance of DNA markers and contribute to developing high resolution genetic maps suitable for genetic analysis. The aim of this study was to expand the number of Diversity Array Technology (DArT) markers on the wheat array that can be mapped in the wheat genome, and to determine their chromosomal location with respect to simple sequence repeat (SSR) markers and their position on the cytogenetic map. A total of 749 and 512 individual DArT and SSR markers, respectively, were identified on at least one of four genetic maps derived from recombinant inbred line (RIL) or doubled haploid (DH) populations. A number of clustered DArT markers were observed in each genetic map, in which 20–34% of markers were redundant. Segregation distortion of DArT and SSR markers was also observed in each mapping population. Only 14% of markers on the Version 2.0 wheat array were assigned to chromosomal bins by deletion mapping using aneuploid lines. In this regard, methylation effects need to be considered when applying DArT marker in genetic mapping. However, deletion mapping of DArT markers provides a reference to align genetic and cytogenetic maps and estimate the coverage of DNA markers across the wheat genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Body temperature,thermoregulatory behaviour and pelt characteristics of three colour morphs of springbok (Antidorcas marsupialis)

Using intra-abdominal miniature data loggers, we measured core body temperature in female springbok (Antidorcas marsupialis) of three colour morphs (black, normal and white), free-living in the Karoo, South Africa, for one year. During winter, white springbok displayed lower daily minimum body temperatures (37.4 ± 0.5 °C), than both black (38.1 ± 0.3 °C) and normal (38.0 ± 0.6 °C) springbok. During spring, black springbok displayed higher daily maximum body temperatures (40.7 ± 0.1 °C) than both white (40.2 ± 0.2 °C) and normal (40.2 ± 0.2 °C) springbok. These high maximum body temperatures were associated with larger daily amplitudes of nychthemeral rhythm of body temperature (2.0 ± 0.2 °C), than that of white (1.6 ± 0.1 °C) and normal (1.7 ± 0.2 °C) springbok. Biophysical properties of sample springbok pelts were consistent with these patterns, as the black springbok pelt showed lower reflectance in the visible spectral range, and higher heat load from simulated solar radiation, than did the pelts of the other two springbok. Black springbok had lower diurnal activity in winter, consistent with them having to forage less because their metabolic cost of homeothermy was lower, but were disadvantaged in hot periods. White springbok, by contrast, were more protected from solar heat load, but potentially less able to meet the energy cost of homeothermy in winter. Thus energy considerations may underlie the rarity of the springbok colour morphs.     

Effectiveness of repeated autumn and spring fires for understorey restoration in weed‐invaded temperate eucalypt woodlands

Question. Can strategic burning, targeting differing ecological characteristics of native and exotic species, facilitate restoration of native understorey in weed‐invaded temperate grassy eucalypt woodlands? Location. Gippsland Plains, eastern Victoria, Australia. Methods. In a replicated, 5‐year experimental trial, the effects of repeated spring or autumn burning were evaluated for native and exotic plants in a representative, degraded Eucalyptus tereticornis grassy woodland. Treatments aimed to reduce seed banks and modify establishment conditions of exotic annual grasses, and to exhaust vegetative reserves of exotic perennial grasses. Treatments were applied to three grassland patch types, dominated by the native grass Austrodanthonia caespitosa, ubiquitous exotic annuals, or the common exotic perennial grass Paspalum dilatatum. Results. The dominant native grass Austrodanthonia caespitosa and native forbs were resilient to repeated fires, and target exotic annuals and perennials were suppressed differentially by autumn and spring fires. Exotic annuals were also suppressed by drought, reducing the overall treatment effects but indicating important opportunities for restoration. The initially sparse exotic geophyte Romulea rosea increased in cover with fire and the impact of this species on native forbs requires further investigation. There was minimal increase in diversity of subsidiary natives with fire, probably owing to lack of propagules. Conclusions. While fire is often considered to increase ecosystem invasibility, our study showed that strategic use of fire, informed by the relative responses of available native and exotic taxa, is potentially an effective step towards restoration of weed‐invaded temperate eucalypt woodlands.