Design of a Debridement Device Using Impinging Jets

Chronic wound care is a significant burden on the healthcare system, affecting an estimated three to six million Americans, manifesting as ulcers associated with restricted blood flow, diabetes mellitus, or pressure. Treatment is frequently unsuccessful, with only an estimated 25–50% of venous and diabetic ulcers closing after 20 weeks of treatment. Debridement, the removal of necrotic tissue and foreign materials from the wounds, is a crucial component in the chronic wound care. While there exist many debridement techniques, the search for new and more effective methods is ongoing. The existing methods of debridement include surgical, the industry gold standard, as well as the mechanical, autolytic, enzymatic, and hydrosurgery (VersaJet™). The VersaJet™ uses a single high-speed jet directed parallel to the wound surface to remove soft necrotic tissue. This paper presents the design of a debridement device that uses two narrow, high-speed impinging fluid jets to excise necrotic tissue. The handheld device can be used to remove strips of necrotic tissue of a predetermined width and depth and was tested on samples of simulated slough, the soft necrotic tissue, and eschar, the hard, scablike necrotic tissue. The preliminary tests indicate that the technique removes necrotic tissue quickly and with good control, suggesting that, with further development, the technique may provide a time-saving alternative to surgical debridement. Further testing, however, is required to ensure that the jets do not damage the surrounding healthy tissues and to quantitatively analyze the effectiveness of the technique relative to other debridement strategies

Resistance and susceptibility of Phaseolus vulgaris to bacteria

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Prion diseases are efficiently transmitted by blood transfusion in sheep

The emergence of variant Creutzfeld-Jakob disease, following on from the bovine spongiform encephalopathy (BSE) epidemic, led to concerns about the potential risk of iatrogenic transmission of disease by blood transfusion and the introduction of costly control measures to protect blood supplies. We previously reported preliminary data demonstrating the transmission of BSE and natural scrapie by blood transfusion in sheep. The final results of this experiment, reported here, give unexpectedly high transmission rates by transfusion of 36% for BSE and 43% for scrapie. A proportion of BSE-infected tranfusion recipients (3 of 8) survived for up to 7 years without showing clinical signs of disease. The majority of transmissions resulted from blood collected from donors at more than 50% of the estimated incubation period. The high transmission rates and relatively short and consistent incubation periods in clinically positive recipients suggest that infectivity titers in blood were substantial and/or that blood transfusion is an efficient method of transmission. This experiment has established the value of using sheep as a model for studying transmission of variant Creutzfeld-Jakob disease by blood products in humans. (Blood. 2008; 112: 4739-4745

A sensitive 301V BSE serial PMCA assay

The prion strain 301V, is a mouse passaged form of bovine spongiform encephalopathy (BSE). It has been used as a model of BSE for more than 20 years, in particular in the investigation of tissue distribution of infectivity, the molecular phenotype and transmission properties of BSE, strain typing assays and prion inactivation studies. Most 301V experiments have required murine bioassay as a method for the quantitation of infectivity. To date this model strain has not been studied with the protein misfolding cyclic amplification assay (PMCA) which detects prion-associated PrPSc protein. The detection of BSE PrPSc by PMCA can be more sensitive than mouse bioassay and is carried out in a much shorter time frame of days as opposed to months/years. Here, we describe the development of a new highly sensitive and specific PMCA assay for murine 301V and assess the sensitivity of the assay in direct comparison with murine bioassay of the same material. This in vitro assay detected, in a few days, 301V at a brain dilution of at least 1x10-9, compared to bioassay of the same material in VM mice that could detect down to a 1x10-8 dilution and took >180 days. The 301V PMCA may therefore offer a faster and more sensitive alternative to live animal bioassay when studying the BSE agent in VM mice

No evidence of subclinical infection in sheep surviving oral challenge with prions

Variant Creutzfeldt-Jakob disease (vCJD) is a fatal zoonotic disease caused by the ingestion of bovine spongiform encephalopathy (BSE)-infected meat products. Although the number of vCJD cases due to dietary exposure has significantly declined, the true burden of subclinical infections remains uncertain. Several large-scale surveys using appendix tissue samples have indicated the presence of abnormal prion protein (PrP Sc; Sc for scrapie) in lymphoid tissue of a small proportion of the UK population. These may represent silent carriers of infection, with the potential to contribute to transmission, persistence and re-emergence of vCJD. Previously, we showed that subclinical infection is a frequent outcome of low-dose prion exposure by blood transfusion in sheep. To determine whether subclinical infection was also found following low-dose exposure by another clinically relevant route for humans, we screened archived tissues from sheep orally challenged with a range of doses of BSE, which did not show clinical or pathological signs of disease after several years of follow-up post-infection. Using a highly sensitive protein misfolding cyclic amplification assay, we were unable to detect PrP Sc in the lymph node/tonsil of 15 sheep, or in a wider range of lymphoid tissues and brain (medulla oblongata) from a subset of 5 sheep. Our findings suggest that the route of infection/exposure may significantly influence the probability of establishing subclinical infection, with the oral route apparently much less efficient than intravenous infection by blood transfusion in sheep. </p

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo.

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'naïve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments

Recombination rate and selection strength in HIV intra-patient evolution

The evolutionary dynamics of HIV during the chronic phase of infection is driven by the host immune response and by selective pressures exerted through drug treatment. To understand and model the evolution of HIV quantitatively, the parameters governing genetic diversification and the strength of selection need to be known. While mutation rates can be measured in single replication cycles, the relevant effective recombination rate depends on the probability of coinfection of a cell with more than one virus and can only be inferred from population data. However, most population genetic estimators for recombination rates assume absence of selection and are hence of limited applicability to HIV, since positive and purifying selection are important in HIV evolution. Here, we estimate the rate of recombination and the distribution of selection coefficients from time-resolved sequence data tracking the evolution of HIV within single patients. By examining temporal changes in the genetic composition of the population, we estimate the effective recombination to be r=1.4e-5 recombinations per site and generation. Furthermore, we provide evidence that selection coefficients of at least 15% of the observed non-synonymous polymorphisms exceed 0.8% per generation. These results provide a basis for a more detailed understanding of the evolution of HIV. A particularly interesting case is evolution in response to drug treatment, where recombination can facilitate the rapid acquisition of multiple resistance mutations. With the methods developed here, more precise and more detailed studies will be possible, as soon as data with higher time resolution and greater sample sizes is available.Comment: to appear in PLoS Computational Biolog

All clinically-relevant blood components transmit prion disease following a single blood transfusion: a sheep model of vCJD

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion