Potential role of pre-existing blood vessels for vascularization and mineralization of osteochondral grafts: an intravital microscopic study in mice

BACKGROUND: The aim of this study was to develop an experimental model that allows to elude the potential role of the preexisting graft microvasculature for vascularization and mineralization of osteochondral grafts. ANIMALS AND METHODS: For that purpose, the II-IV metatarsals of fetal DDY-mice known to be nonvascularized at day 16 of gestation (M16) but vascularized at day 18 (M18) were freshly transplanted into dorsal skin fold chambers of adult DDY mice. Using intravital microscopy angiogenesis, leukocyte-endothelium interaction and mineralization were assessed for 12 days. RESULTS: Angiogenesis occurred at 32 hours in M18, but not before 57 hours in M16 (p = 0.002), with perfusion of these vessels at 42 hours (p = 0.005) and 65 hours (p = 0.1), respectively. Vessels reached a density three times as high as that of the recipient site at day 6, remaining constant until day 12 in M18, whereas in M16 vascular density increased from day 6 and reached that of M18 at day 12 (p = 0.04). Leukocyte-endothelium interaction showed sticker counts elevated by a factor of 4-5 in M18 as compared to M16. Mineralization of osteochondral grafts did not differ between M16 and M18, which significantly increased in both groups throughout the observation period. INTERPRETATION: We propose the faster kinetics in the angiogenic response to M18 and the elevated counts of sticking leukocytes to rest on the potential of establishing end-to-end anastomoses (inosculation) of the vascularized graft with recipient vessels.

Functions of lipid raft membrane microdomains at the blood-brain barrier

The blood-brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).

Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of ICAM-2, JAM-A, and PECAM-1

Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.

Effects of HIV type-1 immune selection on susceptability to integrase inhibitor resistance

BACKGROUND: All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1 integrase sequences from 342 antiretroviral-naive individuals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. METHODS: Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8(+) T-cell escape mutations. RESULTS: In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. CONCLUSIONS: Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.

Hepatitis C virus drug resistance and immune-driven adaptations: relevance to new antiviral therapy

The efficacy of specifically targeted anti-viral therapy for hepatitis C virus (HCV) (STAT-C), including HCV protease and polymerase inhibitors, is limited by the presence of drug-specific viral resistance mutations within the targeted proteins. Genetic diversity within these viral proteins also evolves under selective pressures provided by host human leukocyte antigen (HLA)-restricted immune responses, which may therefore influence STAT-C treatment response. Here, the prevalence of drug resistance mutations relevant to 27 developmental STAT-C drugs, and the potential for drug and immune selective pressures to intersect at sites along the HCV genome, is explored. HCV nonstructural (NS) 3 protease or NS5B polymerase sequences and HLA assignment were obtained from study populations from Australia, Switzerland, and the United Kingdom. Four hundred five treatment-naïve individuals with chronic HCV infection were considered (259 genotype 1, 146 genotype 3), of which 38.5% were coinfected with human immunodeficiency virus (HIV). We identified preexisting STAT-C drug resistance mutations in sequences from this large cohort. The frequency of the variations varied according to individual STAT-C drug and HCV genotype/subtype. Of individuals infected with subtype 1a, 21.5% exhibited genetic variation at a known drug resistance site. Furthermore, we identified areas in HCV protease and polymerase that are under both potential HLA-driven pressure and therapy selection and identified six HLA-associated polymorphisms (P

Host genetic determinants of spontaneous hepatitis C clearance

Acute infection with the hepatitis C virus (HCV) induces a wide range of innate and adaptive immune responses. A total of 20-50% of acutely HCV-infected individuals permanently control the virus, referred to as 'spontaneous hepatitis C clearance', while the infection progresses to chronic hepatitis C in the majority of cases. Numerous studies have examined host genetic determinants of hepatitis C infection outcome and revealed the influence of genetic polymorphisms of human leukocyte antigens, killer immunoglobulin-like receptors, chemokines, interleukins and interferon-stimulated genes on spontaneous hepatitis C clearance. However, most genetic associations were not confirmed in independent cohorts, revealed opposing results in diverse populations or were limited by varying definitions of hepatitis C outcomes or small sample size. Coordinated efforts are needed in the search for key genetic determinants of spontaneous hepatitis C clearance that include well-conducted candidate genetic and genome-wide association studies, direct sequencing and follow-up functional studies.

Cerebral extension of steroid-responsive meningitis arteritis in a boxer

A comatose 30-month-old, entire male boxer was presented because of an acute history of a cluster of three to four seizures. Neurological examination suggested a diffuse to multifocal intracranial lesion. Magnetic resonance tomography revealed symmetrical multifocal to diffuse changes of the cerebral grey matter and ependymal lining with sediment in the lateral ventricles. Haematological examination revealed leucocytosis with neutrophilia. Cerebrospinal fluid examination revealed high protein concentration and polymorphonuclear pleocytosis. Despite antiepileptic treatment, therapy against increased intracranial pressure and antibiosis, the dog's condition continued to deteriorate and he was euthanased. Pathological examination revealed fibrinosuppurative meningo-ependymitis and necrotising arteritis throughout the brain. In addition, chronic inflammation and arterial stenosis was found in the spinal meninges. No infectious agent was found. A diagnosis of steroid-responsive meningitis arteritis was made. The massive extension into the meninges and ventricular system of the forebrain has not been described previously in dogs with steroid-responsive meningitis arteritis and should be considered in the differential diagnosis when an intracranial suppurative infection is suspected.

Gene expression profiling in anti-CD11d mAb-treated spinal cord-injured rats

Acute administration of a mononclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. We have profiled gene expression in anti-CD11d and isotyped-matched control mAb-treated rats after SCI. Microarray analysis demonstrated reduced expression of pro-inflammatory cytokines and increased expression of inflammatory mediators that promote wound healing and the expression of scar proteins predicted to improve nerve growth. These changes in gene expression may reflect changes in the types of macrophages that populate the lesions in anti-CD11d mAb-treated rats.

In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8

Background: Basophils constitute a rare leukocyte population known for their effector functions in inflammation and allergy, as well as more recently described immunoregulatory roles. Besides their low frequency, functional analysis of basophils is hindered by a short life span, inefficient ex vivo differentiation protocols, and lack of suitable cell models. A method to produce large quantities of basophils in vitro would facilitate basophil research and constitute a sought-after tool for diagnostic and drug testing purposes. Methods: A method is described to massively expand bone marrow–derived basophils in vitro. Myeloid progenitors are conditionally immortalized using Hoxb8 in the presence of interleukin-3 (IL-3) and outgrowing cell lines selected for their potential to differentiate into basophils upon shutdown of Hoxb8 expression. Results: IL-3-dependent, conditional Hoxb8-immortalized progenitor cell lines can be expanded and maintained in culture for prolonged periods. Upon shutdown of Hoxb8 expression, near-unlimited numbers of mature functional basophils can be differentiated in vitro within six days. The cells are end-differentiated and short-lived and express basophil-specific surface markers and proteases. Upon IgE- as well as C5a-mediated activation, differentiated basophils release granule enzymes and histamine and secrete Th2-type cytokines (IL-4, IL-13) and leukotriene C4. IL-3-deprivation induces apoptosis correlating with upregulation of the BH3-only proteins BCL-2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) and downregulation of proviral integration site for Moloney murine leukemia virus 1 kinase (PIM-1). Conclusion: A novel method is presented to generate quantitative amounts of mouse basophils in vitro, which moreover allows genetic manipulation of conditionally immortalized progenitors. This approach may represent a useful alternative method to isolating primary basophils.

Gingival fluid cytokine expression and subgingival bacterial counts during pregnancy and postpartum: a case series

OBJECTIVES The aim of this study was to assess gingival fluid (GCF) cytokine messenger RNA (mRNA) levels, subgingival bacteria, and clinical periodontal conditions during a normal pregnancy to postpartum. MATERIALS AND METHODS Subgingival bacterial samples were analyzed with the checkerboard DNA-DNA hybridization method. GCF samples were assessed with real-time PCR including five proinflammatory cytokines and secretory leukocyte protease inhibitor. RESULTS Nineteen pregnant women with a mean age of 32 years (S.D. ± 4 years, range 26-42) participated in the study. Full-mouth bleeding scores (BOP) decreased from an average of 41.2% (S.D. ± 18.6%) at the 12th week of pregnancy to 26.6% (S.D. ± 14.4%) at the 4-6 weeks postpartum (p < 0.001). Between week 12 and 4-6 weeks postpartum, the mean probing pocket depth changed from 2.4 mm (S.D. ± 0.4) to 2.3 mm (S.D. ± 0.3) (p = 0.34). Higher counts of Eubacterium saburreum, Parvimonas micra, Selenomonas noxia, and Staphylococcus aureus were found at week 12 of pregnancy than at the 4-6 weeks postpartum examinations (p < 0.001). During and after pregnancy, statistically significant correlations between BOP scores and bacterial counts were observed. BOP scores and GCF levels of selected cytokines were not related to each other and no differences in GCF levels of the cytokines were observed between samples from the 12th week of pregnancy to 4-6 weeks postpartum. Decreasing postpartum counts of Porphyromonas endodontalis and Pseudomonas aeruginosa were associated with decreasing levels of Il-8 and Il-1β. CONCLUSIONS BOP decreased after pregnancy without any active periodontal therapy. Associations between bacterial counts and cytokine levels varied greatly in pregnant women with gingivitis and a normal pregnancy outcome. Postpartum associations between GCF cytokines and bacterial counts were more consistent. CLINICAL RELEVANCE Combined assessments of gingival fluid cytokines and subgingival bacteria may provide important information on host response.

Associations of Forest Type, Parasitism and Body Condition of Two European Passerines, Fringilla coelebs and Sylvia atricapilla

Human-induced forest modification can alter parasite-host interactions and might change the persistence of host populations. We captured individuals of two widespread European passerines (Fringilla coelebs and Sylvia atricapilla) in southwestern Germany to disentangle the associations of forest types and parasitism by haemosporidian parasites on the body condition of birds. We compared parasite prevalence and parasite intensity, fluctuating asymmetries, leukocyte numbers, and the heterophil to lymphocyte ratio (H/L-ratio) among individuals from beech, mixed-deciduous and spruce forest stands. Based on the biology of bird species, we expected to find fewer infected individuals in beech or mixed-deciduous than in spruce forest stands. We found the highest parasite prevalence and intensity in beech forests for F. coelebs. Although, we found the highest prevalence in spruce forests for S. atricapilla, the highest intensity was detected in beech forests, partially supporting our hypothesis. Other body condition or health status metrics, such as the heterophil to lymphocyte ratio (H/L-ratio), revealed only slight differences between bird populations inhabiting the three different forest types, with the highest values in spruce for F. coelebs and in mixed-deciduous forests for S. atricapilla. A comparison of parasitized versus non-parasitized individuals suggests that parasite infection increased the immune response of a bird, which was detectable as high H/L-ratio. Higher infections with blood parasites for S. atricapilla in spruce forest indicate that this forest type might be a less suitable habitat than beech and mixed-deciduous forests, whereas beech forests seem to be a suboptimal habitat regarding parasitism for F. coelebs.

DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

Hijacking of Host Cell Signaling by Theileria

The apicomplexan parasites Theileria annulata and T. parva possess the ability to transform the infected host cell and induce uncontrolled proliferation. Residing free in the cytosol of its host leukocyte, the schizont is in a perfect position to manipulate host cell signaling pathways involved in regulating apoptosis, proliferation, and cell motility. While extensive Theileria-induced changes in host cell protein phosphorylation patterns have been reported, no Theileria-encoded kinases or phosphatases have been demonstrated - or are even predicted - to be associated with the schizont surface or secreted into the host cell. Instead, it seems that Theileria has evolved the capacity to modulate kinases of the host cell. In certain cases this involves “hijacking” pivotal kinases, such as the IκB kinase complex or the mitotic kinase polo-like kinase 1, recruiting them to the schizont surface. In this chapter the current understanding of Theileria-induced changes in host cell kinase activation is reviewed, and an attempt is made to link these events to phenotypic changes that occur in the cell in response to Theileria infection.