A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.     

An Unusual Genomic Position Effect on Drosophila White Gene Expression: Pairing Dependence, Interactions with Zeste, and Molecular Analysis of Revertants

The white gene in the AR4-24 P[white,rosy] insertion on chromosome 2 has a novel expression pattern, in which it is repressed in the dorsal half of the eye. X-ray mutagenesis led to the isolation of six revertants mapping to chromosome 2, which are wild type in a zeste+ background, and three extreme derivatives, in which white gene expression is repressed in ventral regions of the eye as well. By Southern blot analyses the breakpoints of five of the revertants and one of the extreme derivatives were mapped in the flanking DNA bordering each side of the AR4-24 insertion. The revertants show some dorsal repression of white in the presence of z1, and by this criterion each is only a partial revertant. The extreme derivatives act not only in cis, but also in trans to repress expression of AR4-24 and its various derivatives. We provide evidence that these trans effects are proximity-dependent effects, possibly mediated by pairing of gene copies, as they do not extend to copies of the white gene located elsewhere in the genome. We show that one extreme derivative, E1, is a small deletion spanning the insertion site at the 5' end of the white gene, and propose that the distance between a negative regulatory element in the 5' flanking DNA and the white promoter influences the degree of the repression.     

Multimodal assessment of pain in the esophagus: a new experimental model

A new multimodal pain assessment model was developed integrating electrical, mechanical, cold, and warmth stimuli into the same device. The device, with a bag and electrodes for electrical stimulation, was positioned in the lower part of the esophagus in 11 healthy subjects. Mechanical stimuli were delivered with an impedance planimetric system. Thermal stimuli were performed by circulating water of different temperatures (5-50 degrees C) inside the bag. All subjects reported both nonpainful and painful local and referred sensations to all stimuli. Temporal summation to repeated electrical stimuli could be studied. For all stimuli, there was a relationship between stimulus intensity and pain intensity. The referred pain area increased with increasing intensity of the electrical and mechanical stimuli. There were several differences between the sensations evoked by the four stimulus modalities, indicating activation of different visceral nerve pathways. This model offers the possibility for controlled multimodal stimuli activating the superficial and deeper layers of the human gut and should be used in basic, clinical, and pharmacological pain studies.     

Ruthenium(II) complexes possessing the η-p-cymene ligand

Complexes possessing a soft donor η⁶-arene and hard donor acetylacetonate ligand, [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1) (OTf = trifluoromethanesulfonate; acac = acetylacetonate) and {Ar′ = 3,5-(CF₃)-C₆H₃}, were prepared and fully characterized. The lability of the μ-CH linkage for complex 1 and the THF ligand of 2 allow access to the unsaturated cation [(η⁶-p-cymene)Ru(κ²-O,O-acac)]⁺. The reaction of with KTp {Tp = hydridotris(pyrazolyl)borate} produces . The azide complex forms upon reaction of with N₃Ar (Ar = p-tolyl), and reaction of with CHCl₃ at 100 °C yields the chloride-bridged binuclear complex . The details of solid-state structures of [(η⁶-p-cymene)Ru(κ²-O,O-acac-μ-CH)]₂[OTf]₂ (1), and are disclosed.     

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.     

Expression of proteinase-activated receptor 1-4 (PAR 1-4) in human cancer

Proteinase activated receptors (PAR 1-4) are membrane receptors with a unique way of activation by proteinases like thrombin, trypsin and matrix metalloproteinases which lead to a specific cellular response. To evaluate the significance of expression and co-expression of PAR in cancer we performed a survey on published data. A Pubmed literature search on “PAR, thrombin, cancer” was performed and 46 publications were selected for systematic review based on the availability of information on tumor type, material type, detection method and specification of positive cases. PAR-1 was found in 77.3% of malignant samples (n = 678), PAR-2 in 79.5% (n = 592), PAR-3 in 12.6% (n = 87) and PAR-4 in 54.9% (n = 153). PAR-1 and -2 were present in adenocarcinomas, melanomas, osteosarcomas, glioblastomas, meningiomas, leukaemias and squamous cell carcinomas. Presence of PAR-3 was limited to kidney and liver cancer. The data on PAR-4 expression was inconclusive. Those studies analysing PAR-1 and PAR-2 reported coexpression of the two receptors. PAR-1 and -2 are widely expressed in human tumors suggesting an important role in tumorigenesis and providing potential targets for therapy. PAR-3 and PAR-4 are less frequently detectable, their expression and potential role in tumorigenesis require further investigation.     

Therapeutic window of an EpCAM/CD3-specific BiTE antibody in mice is determined by a subpopulation of EpCAM-expressing lymphocytes that is absent in humans

MuS110 is a BiTE antibody bispecific for murine EpCAM (CD326) and murine CD3. A recent study has shown that muS110 has significant anti tumor activity at well-tolerated doses as low as 5 μg/kg in orthotopic breast and lung cancer models (Amann et al. in Cancer Res 68:143–151, 2008). Here, we have explored the safety profile of muS110 at higher doses. Escalation to 50 μg/kg muS110 caused in mice transient loss of body weight, and transient piloerection, hypomotility, hypothermia and diarrhoea. These clinical signs coincided with serum peaks of TNF-α, IL-6, IL-2, IFN-γ and IL-4, and an increase of surface markers for T cell activation. Because activation of T cells in response to BiTE antibodies is typically dependent on target cells, we analyzed mouse blood for the presence of EpCAM⁺ cells. Various mouse strains presented with a subpopulation of 2–3% EpCAM⁺ blood cells, mostly B and T lymphocytes, which was not detected in human blood samples. In vitro experiments in which the number of EpCAM⁺ cells in blood samples was either reduced or increased suggested that both T cell activation and cytokine release in response to muS110 was dependent on the number of target-expressing cells. In support for a role of EpCAM⁺ lymphocytes in the observed side effects, reduction of EpCAM⁺ blood cells in mice via a low-dose pre treatment with muS110 dramatically increased the tolerability of animals up to at least 500 μg/kg of the BiTE antibody. This high tolerability to muS110 occurred in the presence of non-compromised T cells. No damage to EpCAM⁺ epithelial tissues was evident from histopathological examination of animals daily injected with 100 μg/kg muS110 for 28 days. In summary, these observations suggest that side effects of muS110 in mice were largely caused by an acute T cell activation that was triggered by a subpopulation of EpCAM⁺ lymphocytes. Because humans have extremely low numbers of EpCAM⁺ cells in blood, this toxicity of an EpCAM-specific BiTE may be specific for mice. M. Amann and M. Friedrich contributed equally to this work.