Granulocyte colony-stimulating factor prior to nonmyeloablative irradiation decreases murine host hematopoietic stem cell function and increases engraftment of donor marrow cells

The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation-based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony-stimulating factor (G-CSF) prior to low-dose total body irradiation (LD-TBI) enhanced long-term engraftment of freshly isolated congenic marrow 1.5- to 2-fold more than treatment with LD-TBI alone. This combined regimen was also evaluated in a mouse model of X-linked chronic granulomatous disease (X-CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91(phox) subunit. Long-term engraftment of male X-CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91(phox) was enhanced by approximately 40% when female X-CGD recipients were pretreated with G-CSF prior to 300 cGy. These data confirm that sequential treatment with G-CSF and LD-TBI prior to transplantation increases long-term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G-CSF prior to LD-TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long-term repopulating cells in recipient marrow compared with LD-TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells. Disclosure of potential conflicts of interest is found at the end of this article.     

PCR-based diagnosis of acute and chronic cutaneous leishmaniasis caused by Leishmania (Viannia)

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where Leishmania (Viannia) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the Leishmania subgenus Viannia from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (P < 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (P > 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.     

Emerging extended-spectrum beta-lactamases in Proteus mirabilis

Beta-lactamase production was detected in 147 (52%) of 282 consecutive nonduplicate Proteus mirabilis isolates obtained over a 1-year period from the S. Matteo Hospital of Pavia (northern Italy). Seventy isolates (48% of the beta-lactamase producers) were found to produce extended-spectrum beta-lactamases (ESBLs), identified as PER-1 (first report in this species) and TEM-52 in 52 and 18 isolates, respectively. Analysis of clonal diversity of the ESBL producers suggested different spreading patterns for the two ESBL determinants.     

Oral rapamycin reduces tumour burden and vascularization in Lkb1+/− mice

Patients with Peutz–Jeghers syndrome (PJS) are affected by hamartomatous intestinal polyposis and increased risk of cancers in multiple organs caused by germline mutations in the tumour suppressor gene LKB1. Murine models that recapitulate aspects of PJS have been created. Here we examine the therapeutic effect of rapamycin, a macrolide with anti‐tumourigenic and anti‐angiogenic properties, in reducing tumour incidence in a large cohort of Lkb1^+/? mice. To study the influence of early intervention, the animals were dosed with rapamycin from the age of 8 weeks, well before the onset of polyposis. These mice continued to receive the drug, which was well tolerated, throughout their lives. At sacrifice, we observed a reduction in gastric tumour burden in the rapamycin‐treated mice (p = 0.0001) compared with age‐ and sex‐matched controls. Treated animals also have a lower number of polyps per mouse than controls. In the polyps from the treated mice, phosphorylation of ribosomal p70 S6 kinase was maintained, while the phosphorylation of AKT at serine‐473 was elevated, suggesting that mTORC1 function is maintained at this dosage. Despite this, a significant reduction in microvessel density was seen in polyps from the rapamycin‐treated mice compared to those from the control mice (p = 5 × 10^?5), suggesting that the anti‐angiogenic effect of rapamycin played a role in polyp reduction. Overall, we demonstrated that prolonged oral administration of rapamycin from an early age is effective in lowering tumour burden in the Lkb1^+/? mice without evident side effects. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Caveolin-1 Expression Determines the Route of Neutrophil Extravasation through Skin Microvasculature

Interleukin-8 plays a key role in the acute inflammatory response by mediating recruitment of neutrophils through vessel walls into affected tissues. During this process, molecular signals guide circulating blood neutrophils to target specific vessels for extravasation and to migrate through such vessels via particular routes. Our results show that levels of endothelial caveolin-1, the protein responsible for the induction of the membrane domains known as caveolae, are critical to each of these processes. We demonstrate that, in response to the intradermal injection of interleukin-8, neutrophils are preferentially recruited to a unique subset of venules that express high levels of intercellular adhesion molecule-1 and low levels of caveolin-1. Our results show that neutrophils traverse human dermal microvascular endothelial cells using one of two pathways: a transcellular route directly through the cell or a paracellular route through cellular junctions. Caveolin-1 expression appears to favor the transcellular path while down-regulation of caveolin-1 promotes the paracellular route.Wounding of the epithelium and entry of a foreign body elicit a series of responses from the innate immune system. One of the main hallmarks of acute inflammation is neutrophil infiltration at the affected site.^1,2 In response to injury or infection, resident phagocytic cells become activated and release inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-8. TNF-α activates the vascular endothelium causing vasodilation and cellular infiltration.³ IL-8 functions as a critical chemotactic factor attracting neutrophils from the blood to the affected area.^1,4It is currently thought that leukocyte recruitment and migration through the vasculature is an active process not only for migrating blood cells but also for endothelial cells lining the vessels. Initially, inflammatory cytokines or bacterial endotoxins induce expression of P- and E-selectin on the surface of microvascular endothelial cells.^5,6 These molecules recognize carbohydrate counterligands on the surface of circulating leukocytes and mediate the tethering and rolling of these cells along vessel walls.^5,6,7 Firm adhesion is then initiated through the upregulation of endothelial adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1, which bind to integrins expressed on the leukocyte surface.^5,7,8 Finally, the leukocyte is induced to migrate through the vessel in a process known as diapedesis.^5,6,7Among the many proteins implicated in the process of diapedesis, the adhesion molecule ICAM-1, which is up-regulated on activated endothelium, and caveolin-1, which is expressed on most terminally differentiated cell types but is largely undetectable in white blood cells, have been most closely associated with the route of transendothelial migration in in vitro systems.^7,9,10 A recent study by Millan et al clearly demonstrates that ICAM-1 and caveolin-1 are involved in directing the path of T lymphoblast migration through human umbilical vein endothelial cells (HUVECs).⁷Although both caveolin-1 and ICAM-1 have been associated with leukocyte transendothelial migration in vitro, the distribution of these proteins in vessels used by migrating leukocytes in vivo remain unclear. While all endothelial cells (ECs) share common features, the vascular tree is known to be extremely heterogeneous. As a result, the precise molecular profile of selectins and adhesion molecules defining vessels targeted for extravasation by circulating leukocytes is unknown. Furthermore, since the phenotype of vessel ECs is determined in large part by their unique in vivo microenvironment, site specific and regional differences in the expression of molecules contributing to the regulation of leukocyte transmigration have yet to be thoroughly characterized.^11,12 In this study, we have examined the in vivo molecular profile of vessels targeted by circulating neutrophils in response to IL-8 in the skin and have determined the effect of the expression of these factors on the route of neutrophil transmigration in vitro.     

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.     

Disturbed homeostasis of lung intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 during sepsis

Cecal ligation and puncture (CLP)-induced sepsis in mice was associated with perturbations in vascular adhesion molecules. In CLP mice, lung vascular binding of (125)I-monoclonal antibodies to intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 revealed sharp increases in binding of anti-ICAM-1 and significantly reduced binding of anti-VCAM-1. In whole lung homogenates, intense ICAM-1 up-regulation was found (both in mRNA and in protein levels) during sepsis, whereas very little increase in VCAM-1 could be measured although some increased mRNA was found. During CLP soluble VCAM-1 (sVCAM-1) and soluble ICAM-1 (sICAM-1) appeared in the serum. When mouse dermal microvascular endothelial cells (MDMECs) were incubated with serum from CLP mice, constitutive endothelial VCAM-1 fell in association with the appearance of sVCAM-1 in the supernatant fluids. Under the same conditions, ICAM-1 cell content increased in MDMECs. When MDMECs were evaluated for leukocyte adhesion, exposure to CLP serum caused increased adhesion of neutrophils and decreased adhesion of macrophages and T cells. The progressive build-up in lung myeloperoxidase after CLP was ICAM-1-dependent and independent of VLA-4 and VCAM-1. These data suggest that sepsis disturbs endothelial homeostasis, greatly favoring neutrophil adhesion in the lung microvasculature, thereby putting the lung at increased risk of injury.     

Insights into Severe 5,10‐Methylenetetrahydrofolate Reductase Deficiency: Molecular Genetic and Enzymatic Characterization of 76 Patients

5,10‐Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%–42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide‐responsiveness, and 24 abnormal kinetics of S‐adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N‐terminal catalytic domain, whereas missense mutations in the C‐terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S‐adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.     

Trans‐Ethnic Meta‐Analysis Identifies Common and Rare Variants Associated with Hepatocyte Growth Factor Levels in the Multi‐Ethnic Study of Atherosclerosis (MESA)

Hepatocyte growth factor (HGF) is a mesenchyme‐derived pleiotropic factor that regulates cell growth, motility, mitogenesis, and morphogenesis in a variety of cells, and increased serum levels of HGF have been linked to a number of clinical and subclinical cardiovascular disease phenotypes. However, little is currently known regarding which genetic factors influence HGF levels, despite evidence of substantial genetic contributions to HGF variation. Based upon ethnicity‐stratified single‐variant association analysis and trans‐ethnic meta‐analysis of 6201 participants of the Multi‐Ethnic Study of Atherosclerosis (MESA), we discovered five statistically significant common and low‐frequency variants: HGF missense polymorphism rs5745687 (p.E299K) as well as four variants (rs16844364, rs4690098, rs114303452, rs3748034) within or in proximity to HGFAC. We also identified two significant ethnicity‐specific gene‐level associations (A1BG in African Americans; FASN in Chinese Americans) based upon low‐frequency/rare variants, while meta‐analysis of gene‐level results identified a significant association for HGFAC. However, identified single‐variant associations explained modest proportions of the total trait variation and were not significantly associated with coronary artery calcium or coronary heart disease. Our findings indicate that genetic factors influencing circulating HGF levels may be complex and ethnically diverse.