Gene replacement with the human BRCA1 locus: tissue specific expression and rescue of embryonic lethality in mice

We have generated transgenic mice that harbor a 140 kb genomic fragment of the human BRCA1 locus (TgN.BRCA1(GEN)). We find that the transgene directs appropriate expression of human BRCA1 transcripts in multiple mouse tissues, and that human BRCA1 protein is expressed and stabilized following exposure to DIVA damage, Such mice are completely normal, with no overt signs of BRCA1 toxicity commonly observed when BRCA1 is expressed from heterologous promoters. Most importantly, however, the transgene rescues the otherwise lethal phenotype associated with the targeted hypomorphic allele (Brca1(Delta exIISA)). Brca1(-/-); TgN.BRCA1(GEN) bigenic animals develop normally and can be maintained as a distinct line. These results show that a 140 kb fragment of chromosome 17 contains all elements necessary for the correct expression, localization, and function of the BRCA1 protein, Further, the model provides evidence that function and regulation of the human BRCA1 gene can be studied and manipulated in a genetically tractable mammalian system.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

An assumption-driven case-specific model editor

A case sensitive intelligent model editor has been developed for constructing consistent lumped dynamic process models and for simplifying them using modelling assumptions. The approach is based on a systematic assumption-driven modelling procedure and on the syntax and semantics of process,models and the simplifying assumptions.

A multi-sectorial committee in directing HIV/AIDS-specific interventions in the occupational setting: An example from South Africa

We present a descriptive analysis of a mechanism to coordinate and implement human immunodeficiency virus (HIV) prevention and care in the occupational setting. The mechanism we describe is a multidisciplinary committee composed of stakeholders in the occupational health environment including unions, management, medical researchers, and medical personnel. The site chosen for the analysis was a South African sugar mill in rural KwaZulu-Natal. The factory is situated in an area of high HIV seroprevalence and has a workforce of 400 employees. The committee was initiated to coordinate a combined prevention-care initiative. The issues that were important in the formation of the committee included confidentiality, trust, and the traditional roles of the stakeholder relationships. When these points were addressed through the focus on a common goal, the committee was able to function in its role as a coordinating body. Central to this success was the inclusion of all stakeholders in the process, including those with traditionally opposing, interests and legitimacy conferred by the stakeholders. This committee was functionally effective and demonstrated the benefit of a freestanding committee dedicated to addressing HIV/acquired immune deficiency syndrome (AIDS) issues. We describe the implementation and feasibility of a multisectoral committee in directing HIV/AIDS initiatives in the occupational setting in rural South Africa.

Pathway-specific targeting of GAB(A) a receptor subtypes to somatic and dendritic synapses in the central amygdala

Neurons in the central amygdala express two distinct types of ionotropic GABA receptor. One is the classical GABA(A) receptor that is blocked by low concentrations of bicuculline and positively modulated by benzodiazepines. The other is a novel type of ionotropic GABA receptor that is less sensitive to bicuculline but blocked by the GABA(C) receptor antagonist (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA) and by benzodiazepines. In this study, we examine the distribution of these two receptor types. Recordings of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) showed a wide variation in amplitude. Most events had amplitudes of 100 pA. Large-amplitude events also had rise times faster than small-amplitude events. Large-amplitude events were fully blocked by 10 muM bicuculline but unaffected by TPMPA. Small amplitude events were partially blocked by both bicuculline and TPMPA. Focal application of hypertonic sucrose to the soma evoked large-amplitude mIPSCs, whereas focal dendritic application of sucrose evoked small-amplitude mIPSCs. Thus inhibitory synapses on the dendrites of neurons in the central amygdala express both types of GABA receptor, but somatic synapses expressed purely GABA(A) receptors. Minimal stimulation revealed that inhibitory inputs arising from the laterally located intercalated cells innervate dendritic synapses, whereas inhibitory inputs of medial origin innervated somatic inhibitory synapses. These results show that different types of ionotropic GABA receptors are targeted to spatially and functionally distinct synapses. Thus benzodiazepines will have different modulatory effects on different inhibitory pathways in the central amygdala.

Lentiviral vector-mediated tyro sinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR'CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4(+) and CD8(+) cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonbese diabetic (NOD) mice, recombinant congenic nonbese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [H-3]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-gencrative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.

CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-kappa B

An understanding of the biochemical control of dendritic cell (DC) differentiation/activation is essential for improving T cell immunity by various immunotherapeutic approaches, including DC immunization. Ligation of CD40 enhances DC function, including conditioning for CTL priming. NF-kappaB, and particularly RelB, is an essential control pathway for myeloid DC differentiation. Furthermore, RelB regulates B cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L) and TNF-alpha, which differ in their capacity to condition DC, would also differ in their capacity to activate NF-kappaB. DC differentiated for 2 days from monocytes in the presence of GM-CSF and IL-4 were used as a model, as NF-kappaB activity was constitutively low. The capacity of DC to activate T cells following CD40L treatment was enhanced compared with TNF-alpha treatment, and this was NF-kappaB dependent. Whereas RelB/p50 translocation induced by TNF-alpha was attenuated after 6 h, RelB/p50 nuclear translocation induced by CD40L was sustained for at least 24 h. The mechanism of this difference related to enhanced degradation of IkappaBalpha following CD40L stimulation. However, NF-kappaB activation induced by TNF-alpha could be sustained by blocking autocrine IL-10. These data indicate that NF-kappaB activation is essential for T cell activation by DC, and that this function is enhanced if DC NF-kappaB activation is prolonged. Because IL-10 moderates DC NF-kappaB activation by TNF-alpha, sustained NF-kappaB activation can be achieved by blocking IL-10 in the presence of stimuli that induce TNF-alpha.

Prediction of promiscuous peptides that bind HLA class I molecules

Promiscuous T-cell epitopes make ideal targets for vaccine development. We report here a computational system, multipred, for the prediction of peptide binding to the HLA-A2 supertype. It combines a novel representation of peptide/MHC interactions with a hidden Markov model as the prediction algorithm. multipred is both sensitive and specific, and demonstrates high accuracy of peptide-binding predictions for HLA-A*0201, *0204, and *0205 alleles, good accuracy for *0206 allele, and marginal accuracy for *0203 allele. multipred replaces earlier requirements for individual prediction models for each HLA allelic variant and simplifies computational aspects of peptide-binding prediction. Preliminary testing indicates that multipred can predict peptide binding to HLA-A2 supertype molecules with high accuracy, including those allelic variants for which no experimental binding data are currently available.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

The protease inhibitor cystatin C is differentially expressed among dendritic cell populations, but does not control antigen presentation

Dendritic cells (DC) undergo complex developmental changes during maturation. The MHC class H (MHC H) molecules of immature DC accumulate in intracellular compartments, but are expressed at high levels on the plasma membrane upon DC maturation. It has been proposed that the cysteine protease inhibitor cystatin C (CyC) plays a pivotal role in the control of this process by regulating the activity of cathepsin S, a protease involved in removal of the MHC H chaperone E, and hence in the formation of MHC H-peptide complexes. We show that CyC is differentially expressed by mouse DC populations. CD8(+) DC, but not CD4(+) or CD4(-)CD8(-) DC, synthesize CyC, which accumulates in MHC II(+)Lamp(+) compartments. However, II processing and MHC H peptide loading proceeded similarly in all three DC populations. We then analyzed MHC H localization and Ag presentation in CD8(+) DC, bone marrow-derived DC, and spleen-derived DC lines, from CyC-deficient mice. The absence of CyC did not affect the expression, the subcellular distribution, or the formation of peptide-loaded MHC II complexes in any of these DC types, nor the efficiency of presentation of exogenous Ags. Therefore, CyC is neither necessary nor sufficient to control MHC II expression and Ag presentation in DC. Our results also show that CyC expression can differ markedly between closely related cell types, suggesting the existence of hitherto unrecognized mechanisms of control of CyC expression.

Auditory lexical decisions in children with specific language impairment

This study examined spoken-word recognition in children with specific language impairment (SLI) and normally developing children matched separately for age and receptive language ability. Accuracy and reaction times on an auditory lexical decision task were compared. Children with SLI were less accurate than both control groups. Two subgroups of children with SLI, distinguished by performance accuracy only, were identified. One group performed within normal limits, while a second group was significantly less accurate. Children with SLI were not slower than the age-matched controls or language-matched controls. Further, the time taken to detect an auditory signal, make a decision, or initiate a verbal response did not account for the differences between the groups. The findings are interpreted as evidence for language-appropriate processing skills acting upon imprecise or underspecified stored representations.

Cytokine expanded myeloid precursors function as regulatory antigen presenting cells and induce tolerance through IL-10 producing regulatory T cells

The initiation of graft vs. host disease (GVHD) after stem cell transplantation is dependent on direct antigen presentation by host antigen presenting cells (APC) while the effect of indirect antigen presentation by donor APC is unknown. We have studied the role of indirect antigen presentation in allogenic responses by adding populations of cytokine-expanded donor APC to haematopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 L molecule) and G-CSF expanded myeloid DC, plasmacytoid DC and a novel granulocyte-monocyte precursor population (GM) that differentiate into class IIpos, CD80/CD86pos, CD40neg APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells induced transplant tolerance via MHC class II restricted generation of IL-10-secreting regulatory T cells. Thus a population of cytokine expanded granulocyte-monocyte precursors function as regulatory antigen presenting cells, suggesting that G-CSF derivatives may have application in disorders characterised by a loss of self-tolerance.