Refining rodent models of spinal cord injury.

This report was produced by an Expert Working Group (EWG) consisting of UK-based researchers, veterinarians and regulators of animal experiments with specialist knowledge of the use of animal models of spinal cord injury (SCI). It aims to facilitate the implementation of the Three Rs (Replacement, Reduction and Refinement), with an emphasis on refinement. Specific animal welfare issues were identified and discussed, and practical measures proposed, with the aim of reducing animal use and suffering, reducing experimental variability, and increasing translatability within this critically important research field

Deficiency of Leishmania phosphoglycans influences the magnitude but does not affect the quality of secondary (memory) anti-Leishmania immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals

Estimating and projecting HIV prevalence and AIDS deaths in Tanzania using antenatal surveillance data

BACKGROUND: The Estimations and Projections Package (EPP 2005) for HIV/AIDS estimates and projects HIV prevalence, number of people living with HIV and new HIV infections and AIDS cases using antenatal clinic (ANC) surveillance data. The prevalence projection produced by EPP can be transferred to SPECTRUM, a demographic projectionmodel, to calculate the number of AIDS deaths. This paper presents estimates and projections of HIV prevalence, new cases of HIV infections and AIDS deaths in Tanzania between 2001 and 2010 using the EPP 2005 and SPECTRUM soft-wares on ANC data. METHODS: For this study we used; the 1985 – 2004 ANC data set, the 2005 UN population estimates for urban and rural adults, which is based on the 2002 population census, and results of the 2003 Tanzania HIV Indicator Survey. The ANC surveillance sites were categorized into urban and rural areas on the basis of the standard national definitions of urban and rural areas, which led to 40 urban and 35 rural clinic sites. The rural and urban epidemics were run independently by fitting the model to all data and on level fits. RESULTS: The national HIV prevalence increased from 0% in 1981 to a peak of 8.1% in 1995, and gradually decreased to 6.5% in 2004 which stabilized until 2010. The urban HIV epidemic increased from 0% in 1981 peaking at 12.6% in 1992 and leveled to between 10.9% and 11.8% from 2003 to 2010. The rural epidemic peaked in 1995 at 7.0% and gradually declined to 5.2% in 2004, and then stabilized at between 5.1% and 5.3% from 2005 to 2010. New infections are projected to rise steadily, resulting in 250,000 new cases in 2010. Deaths due to AIDS started in 1985 and rose steadily to reach 120,000 deaths in 2010, with more females dying than men. CONCLUSION: The fact that the number of new infections is projected to increase steadily to reach 250,000 per year in 2010 calls for more concerted efforts to combat the spread of HIV infection particularly in the rural areas where the infrastructure needed for prevention programmes such as counseling and testing, condom accessibility and AIDS information is less developed

Heteropolymeric Triplex-Based Genomic Assay® to Detect Pathogens or Single-Nucleotide Polymorphisms in Human Genomic Samples

Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP), without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are “canonical triplexes”. Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays

The concept of "compartment allergy": prilocaine injected into different skin layers

We herein present a patient with delayed-type allergic hypersensitivity against prilocaine leading to spreading eczematous dermatitis after subcutaneous injections for local anesthesia with prilocaine. Prilocaine allergy was proven by positive skin testing and subcutaneous provocation, whereas the evaluation of other local anesthetics - among them lidocaine, articaine and mepivacaine - did not exhibit any evidence for cross-reactivity

Induction of Protective CD4+ T Cell-Mediated Immunity by a Leishmania Peptide Delivered in Recombinant Influenza Viruses

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4+ Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK158–173 CD4+ peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK158–173-specific CD4+ T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK158–173 triggers LACK158–173-specific Th1-biased CD4+ T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2–4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4+ T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK158–173 led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation

Protecting Vulnerable Research Subjects in Critical Care Trials: Enhancing the Informed Consent Process and Recommendations for Safeguards

Although critically ill patients represent a vulnerable group of individuals, guidelines in research ethics assert that ethically acceptable research may proceed with such vulnerable subjects if additional safeguards are in place to minimize the risk of harm and exploitation. Such safeguards include the proper obtainment of informed consent that avoids the presence of the therapeutic misconception and the assessment of decisional capacity in critically ill patients recruited for research. Also discussed in this review are additional safeguards for such vulnerable subjects, as well as the issues involved with proxy consent. Heightened awareness to principles of ethics and provision of additional safeguards to enhance protections of vulnerable subjects would help to maintain the public trust in the research endeavor

Recent breast cancer trends among Asian/Pacific Islander, Hispanic, and African-American women in the US: changes by tumor subtype

Abstract available at publisher's website