Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379.

Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer.

Commonly used disinfectants fail to eradicate Salmonella enterica biofilms from food contact surface materials.

Salmonellosis is the second most common cause of food-borne illness worldwide. Contamination of surfaces in food processing environments may result in biofilm formation with a risk of food contamination. Effective decontamination of biofilm-contaminated surfaces is challenging. Using the CDC biofilm reactor, the activities of sodium hypochlorite, sodium hydroxide, and benzalkonium chloride were examined against an early (48-h) and relatively mature (168-h) Salmonella biofilm. All 3 agents result in reduction in viable counts of Salmonella; however, only sodium hydroxide resulted in eradication of the early biofilm. None of the agents achieved eradication of mature biofilm, even at the 90-min contact time. Studies of activity of chemical disinfection against biofilm should include assessment of activity against mature biofilm. The difficulty of eradication of established Salmonella biofilm serves to emphasize the priority of preventing access of Salmonella to postcook areas of food production facilities.

Salmonella enterica biofilm formation and density in the Centers for Disease Control and Prevention's biofilm reactor model is related to serovar and substratum.

Foodborne pathogens can attach to, and survive on, food contact surfaces for long periods by forming a biofilm. Salmonella enterica is the second most common cause of foodborne illness in Ireland. The ability of S. enterica to form a biofilm could contribute to its persistence in food production areas, leading to cross-contamination of products and surfaces. Arising from a large foodborne outbreak of S. enterica serovar Agona associated with a food manufacturing environment, a hypothesis was formulated that the associated Salmonella Agona strain had an enhanced ability to form a biofilm relative to other S. enterica. To investigate this hypothesis, 12 strains of S. enterica, encompassing three S. enterica serovars, were assessed for the ability to form a biofilm on multiple food contact surfaces. All isolates formed a biofilm on the contact surfaces, and there was no consistent trend for the Salmonella Agona outbreak strain to produce a denser biofilm compared with other strains of Salmonella Agona or Salmonella Typhimurium. However, Salmonella Enteritidis biofilm was considerably less dense than Salmonella Typhimurium and Salmonella Agona biofilms. Biofilm density was greater on tile than on concrete, polycarbonate, stainless steel, or glass.

Haematopoietic stem cell recruitment to injured murine liver sinusoids depends on (alpha)4(beta)1 integrin/VCAM-1 interactions.

OBJECTIVE: Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia-reperfusion (IR) injury and in vitro using flow-based adhesion assays. DESIGN: Partial IR was induced in anaesthetised WT or PECAM-1(-/-) mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against alpha(4)beta(1) integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS. RESULTS: Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-alpha(4)beta(1) and anti-VCAM-1 antibodies. A role for alpha(4)beta(1) was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased alpha(4)beta(1) expression. CD18, CD44 and PECAM-1 were not involved in recruitment. CONCLUSIONS: This novel study demonstrates that the alpha(4)beta(1)/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.

Vitronectin in human hepatic tumours contributes to the recruitment of lymphocytes in an alpha v beta3-independent manner.

The degree of lymphocyte infiltration is a prognostic factor in liver cancer, but to date the mechanisms by which lymphocytes infiltrate into and are retained in hepatic tumours are poorly understood. We hypothesised that the extracellular matrix glycoprotein vitronectin, a major component of the stroma of hepatic tumours, might play a role in the recruitment and retention of tumour-infiltrating lymphocytes (TIL). Thus, we investigated the ability of vitronectin to support migration and adhesion of TIL isolated from hepatocellular carcinoma and colorectal hepatic metastases. Soluble vitronectin-induced dose-dependent migration of TIL in in vitro chemotaxis and haptotaxis assays and vitronectin in tissue sections was able to support TIL adhesion to tumour stroma. Neither adhesion nor migration was inhibited by a function blocking mAb against the major vitronectin receptor alpha v beta3 and we were unable to detect alpha v beta3 on TIL in vitro or in vivo on tumour tissue. However, TIL did express high levels of urokinase-type plasminogen activator receptor (uPAR) and inhibitory antibodies and amiloride both significantly inhibited TIL adhesion to vitronectin and reduced transendothelial migration of lymphocytes across liver endothelium in vitro. Thus, we provide evidence that vitronectin in liver tumours can support the recruitment and retention of effector lymphocytes by an uPAR-dependent mechanism.

Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo.

The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of blood-borne compounds to the parenchyma. They are functionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understanding the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phenotypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

Endothelial interactions of neutrophils under flow in chronic obstructive pulmonary disease.

It is generally accepted that the neutrophil is central to the pathogenesis of chronic obstructive pulmonary disease (COPD). Enhanced endothelial interactions of this cell may contribute to the susceptibility of smokers who develop the disease; however, these interactions have not previously been studied in COPD. The aim of the current study was to determine whether enhanced endothelial interactions of neutrophils from smokers are a predisposing factor for the development of COPD. Endothelial interactions under flow and adhesion molecule expression of peripheral blood neutrophils were compared between seven never-smokers (NS), seven healthy smokers (HS), 11 COPD patients with severe alpha1-antitrypsin deficiency (PiZ) and neutrophils from 11 COPD patients without the deficiency (PiM). Total adhesive and migratory responses (per mm2 endothelium per 10(6) neutrophils) were significantly greater in the PiM group (mean+/-se 704.2+/-57.9 versus 509.3+/-48.8 in the PiZ group, 499.3+/-40.1 in the HS and 491.2+/-33.7 in the NS). This corresponded with increased macrophage antigen-1 (CD11b) expression on stimulated neutrophils in the PiM group compared with the PiZ group (mean+/-se relative fluorescence intensity 1.4+/-0.1 versus 1.1+/-0.1). In conclusion, the enhanced endothelial interaction of neutrophils from smokers who have developed chronic obstructive pulmonary disease in the presence of normal levels of alpha1-antitrypsin deficiency, but not in those with severe alpha(1)-antitrypsin deficiency, suggests that this is a predisposing factor for the development of the disease, and upregulation of macrophage antigen-1 may be responsible.

A cationic lipid-formulated plasmid DNA vaccine confers sustained antibody-mediated protection against aerosolized anthrax spores.

DNA vaccines provide an attractive technology platform against bioterrorism agents due to their safety record in humans and ease of construction, testing, and manufacture. We have designed monovalent and bivalent anthrax plasmid DNA (pDNA) vaccines encoding genetically detoxified protective antigen (PA) and lethal factor (LF) proteins and tested their immunogenicity and ability to protect rabbits from an aerosolized inhalation spore challenge. Immune responses after two or three injections of cationic lipid-formulated PA, PA plus LF, or LF pDNAs were at least equivalent to two doses of anthrax vaccine adsorbed (AVA). High titers of anti-PA, anti-LF, and neutralizing antibody to lethal toxin (Letx) were achieved in all rabbits. Eight or nine animals in each group were challenged with 100x LD(50) of aerosolized anthrax spores 5 or 9 weeks after vaccination. An additional 10 animals vaccinated with PA pDNA were challenged >7 months postvaccination. All animals receiving PA or PA plus LF pDNA vaccines were protected. In addition, 5 of 9 animals receiving LF pDNA survived, and the time to death was significantly delayed in the others. Groups receiving three immunizations with PA or PA plus LF pDNA showed no increase in anti-PA, anti-LF, or Letx neutralizing antibody titers postchallenge, suggesting little or no spore germination. In contrast, titer increases were seen in AVA animals, and in surviving animals vaccinated with LF pDNA alone. Preclinical evaluation of this cationic lipid-formulated bivalent PA and LF vaccine is complete, and the vaccine has received U.S. Food and Drug Administration Investigational New Drug allowance.

Heparin therapy for ulcerative colitis? Effects and mechanisms.

Despite intensive medical treatment with steroids and immunosuppressants, acute colitis is still associated with a colectomy rate of up to 15%. Following the observation that a patient with severe steroid-resistant colitis went into remission when treated with heparin for a deep vein thrombosis, there have been a number of reports on the use of heparin in acute ulcerative colitis. Although small and uncontrolled, these studies consistently demonstrate the beneficial effects of heparin, with surprisingly few side-effects in a disease characterized by mucosal haemorrhage. The mechanisms by which heparin may ameliorate ulcerative colitis remain unclear. A simple anticoagulant effect may be responsible, but similar effects are not seen with warfarin. As a result of their intense negative charge, the glycosaminoglycans that constitute heparin have diverse biological effects. These include potent anti-inflammatory actions, in vitro and in vivo, and the potentiation of the activity of the peptide growth factors necessary for mucosal regeneration and repair. This review summarizes the clinical reports on heparin treatment for ulcerative colitis and explores the mechanisms by which this novel form of treatment may exert its effects.

MAdCAM-1 expressed in chronic inflammatory liver disease supports mucosal lymphocyte adhesion to hepatic endothelium (MAdCAM-1 in chronic inflammatory liver disease).

Mucosal addressin cell adhesion molecule (MAdCAM-1) plays a pivotal role in T-lymphocyte homing to the gut. Given the strong association between the autoimmune liver diseases primary sclerosing cholangitis and autoimmune hepatitis and inflammatory bowel disease, we investigated the role of MAdCAM-1 in recruiting mucosal lymphocytes to the liver. MAdCAM-1 was strongly expressed on inflamed portal vein/sinusoidal endothelium in autoimmune mediated liver disease. In modified Stamper-Woodruff assays, MAdCAM-1 on hepatic vessels supported adhesion of alpha4beta7+ lymphocytes (i.e., gut-derived T cells) from patients with inflammatory bowel disease and primary sclerosing cholangitis. This adhesion was inhibited by pretreatment with blocking antibodies to MAdCAM-1, alpha4beta7, or the integrin alpha4 chain indicating that MAdCAM-1 in inflamed liver is functionally active. Circulating lymphocytes from patients with primary sclerosing cholangitis showed rolling adhesion on MAdCAM-1 transfectants in a flow-based adhesion assay that could be blocked by anti-MAdCAM-1 or anti-alpha4beta7 mAbs. These findings indicate that, under certain circumstances, vessels in the human liver support adhesion of alpha4beta7+ mucosal lymphocytes via binding to aberrantly expressed MAdCAM-1 on liver endothelium. This provides a mechanism to explain the hepatic recruitment of mucosal lymphocytes in inflammatory liver disease complicating inflammatory bowel disease.

Spontaneous repair of full-thickness defects of articular cartilage in a goat model. A preliminary study.

BACKGROUND: Full-thickness defects measuring 3 mm in diameter have been commonly used in studies of rabbits to evaluate new procedures designed to improve the quality of articular cartilage repair. These defects initially heal spontaneously. However, little information is available on the characteristics of repair of larger defects. The objective of the present study was to define the characteristics of repair of 6-mm full-thickness osteochondral defects in the adult Spanish goat. METHODS: Full-thickness osteochondral defects measuring 6 x 6 mm were created in the medial femoral condyle of the knee joint of adult female Spanish goats. The untreated defects were allowed to heal spontaneously. The knee joints were removed, and the defects were examined at ten time-intervals, ranging from time zero (immediately after creation of the defect) to one year postoperatively. The defects were examined grossly, microradiographically, histologically, and with magnetic resonance imaging and computed tomography. RESULTS: The 6-mm osteochondral defects did not heal. Moreover, heretofore undescribed progressive, deleterious changes occurred in the osseous walls of the defect and the articular cartilage surrounding the defect. These changes resulted in a progressive increase in the size of the defect, the formation of a large cavitary lesion, and the collapse of both the surrounding subchondral bone and the articular cartilage into the periphery of the defect. Resorption of the osseous walls of the defect was first noted by one week, and it was associated with extensive osteoclastic activity in the trabecular bone of the walls of the defect. Flattening and deformation of the articular cartilage at the edges of the defect was also observed at this time. By twelve weeks, bone resorption had transformed the surgically created defect into a larger cavitary lesion, and the articular cartilage and subchondral bone surrounding the defect had collapsed into the periphery of the defect. By twenty-six weeks, bone resorption had ceased and the osseous walls of the lesion had become sclerotic. The cavitary lesion did not become filled in with fibrocartilage. Instead, a cystic lesion was found in the center of most of the cavitary lesions. Only a thin layer of fibrocartilage was present on the sclerotic osseous walls of the defect. Specimens examined at one year postoperatively showed similar characteristics. CONCLUSIONS: Full-thickness osteochondral defects, measuring 6 mm in both diameter and depth, that are created in the medial femoral condyle of the knee joint of adult Spanish goats do not heal spontaneously. Instead, they undergo progressive changes resulting in resorption of the osseous walls of the defect, the formation of a large cavitary lesion, and the collapse of the surrounding articular cartilage and subchondral bone. CLINICAL RELEVANCE: As surgeons apply new reparative procedures to larger areas of full-thickness articular cartilage loss, we believe that it is important to consider the potential deleterious effects of a "zone of influence" secondary to the creation of a large defect in the subchondral bone. When biologic and synthetic matrices with or without cells or bioactive factors are placed into surgically created osseous defects, the osseous walls serve as shoulders to protect and stabilize the preliminary repair process. It is important to protect the repair process until biologic incorporation occurs and the chondrogenic switch turns the cells on to synthesize an articular-cartilage-like matrix. It takes a varying period of time to fill a large, surgically created bone defect underlying a chondral surface. The repair of such a defect requires bone synthesis and the reestablishment of a subchondral plate with a tidemark transition to the new overlying articular surface. The prevention of secondary changes in the surrounding bone and articular cartilage and the durability of the new reparative tissue making up the articulating surface are issues that must be addressed in future studies.

Adhesion of lymphocytes to hepatic endothelium.

Chronic inflammation occurs when factors that regulate the process of leucocyte recruitment are disrupted, and it is dependent on recruitment, activation, and retention of lymphocytes within tissue microenvironments. The molecular mechanisms that mediate lymphocyte adhesion to vascular endothelial cells have been described by several groups, but the signals involved in the recruitment of lymphocytes via the hepatic circulation have yet to be elucidated fully. This article considers the liver as a model of organ specific lymphocyte recruitment. In this context, the roles of leucocyte and endothelial adhesion molecules and chemokines in lymphocyte recruitment are discussed. The article also reviews the mechanisms that regulate lymphocyte recirculation to the liver under both physiological and pathological conditions and draws parallels with other organs such as the gut and skin.

Migration of polyethylene particles around stable implants in an animal model.

The aim of this study was to test the hypothesis that a tight seal between bone and implant will eliminate the avenue of particle migration around stable implants. Three types of implants were used in rabbits (polished press-fit Ti-6Al-4V or plasma-sprayed hydroxyapatite [HA]-coated Ti-6Al-4V) or doughy stage polymethyl methacrylate (PMMA). Implants were placed in the condylar notch. Each animal received an intra-articular injection of high density polyethylene (PE) particles (10(8) in 0.4 mL; mean size 4.7 microns) at 4 and 6 weeks postoperatively. Eight weeks postoperatively, peri-implant tissues were examined for PE particles and osteolysis. In all cases, intracellular PE particles were seen at the bone-implant interface and within marrow. No osteolysis was observed. Bone apposition was determined by computerized image analysis. There was no significant difference in the percentage of bone apposition (+/- SD) among the three groups of implants: Ti-6Al-4V (68% +/- 19%), HA-coated Ti-6Al-4V (70% +/- 10%), and PMMA (59% +/- 12%). These results indicate that a polished Ti-6Al-4V surface is as effective as PMMA or HA coating in limiting migration of PE particles around stable osseointegrated implants in rabbits.

Surgical margin in hepatic resection for colorectal metastasis: a critical and improvable determinant of outcome.

OBJECTIVE: To update the analysis of technical and biologic factors related to hepatic resection for colorectal metastasis in a large single-institution series to identify important prognostic indicators and patterns of failure. SUMMARY BACKGROUND DATA: Surgical therapy for colorectal carcinoma metastatic to the liver is the only potentially curable treatment. Careful patient selection of those with resectable liver-only metastatic disease is crucial to the success of surgical therapy. METHODS: Two hundred forty-four consecutive patients undergoing curative hepatic resection for metastatic colorectal carcinoma were analyzed retrospectively. Variables examined included sex, stage of primary lesion, size of liver lesion(s), number of lesions, disease-free interval, ploidy, differentiation, preoperative carcinoembryonic antigen level, and operative factors such as resection margin, use of cryotherapy, intraoperative ultrasound, and blood loss. RESULTS: Surgical margin, number of lesions, and carcinoembryonic antigen (CEA) levels significantly control prognosis. Patients with only one or two liver lesions, a 1-cm surgical margin, and low CEA levels have a 5-year disease-free survival rate of more than 30%. Disease-free interval, original stage, bilobar involvement, size of metastasis, differentiation, and ploidy were not significant predictors of recurrence. The pattern of failure correlates with surgical margin. Routine use of intraoperative ultrasound resulted in an increased incidence of negative surgical margin during the period examined. CONCLUSIONS: Surgical resection or cryotherapy of hepatic metastasis from colorectal cancer is safe and curable in appropriately selected patients. Biologic factors, such as number of lesions and carcinoembryonic antigen levels, determine potential curability, and surgical margin governs the patterns of failure and outcome in potentially curable patients. Optimization of selection criteria and surgical resection margins will improve outcome.

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.

Pathogenicity of Theileria parva is influenced by the host cell type infected by the parasite.

Theileria parva has been shown to infect and transform B cells and T cells at similar frequencies in vitro. However, the majority of parasitized cells in the tissues of infected cattle are alpha/beta T cells. The aim of this study was to determine whether the cell type infected with T. parva influenced the pathogenicity of the parasite. The initial approach, which involved inoculation of cattle with autologous cloned cell lines of different phenotypes, failed to resolve the issue, because of prolonged period of culture required to clone and characterize the cell lines resulted in attenuation of the cells. As an alternative approach, cattle were inoculated with purified populations of autologous cells that had been incubated in vitro with T. parva sporozoites for 48 h. As few as 3 x 10(4) peripheral blood mononuclear cells (PBMC) treated in this way were found to produce severe clinical reactions with high levels of parasitosis. Infections of similar severity were produced with purified populations of CD2+, CD4+, and CD8+ T cells. By contrast, infected B cells gave rise to mild self-limiting infections even when administered at a 10-fold-higher dose. In animals that received infected CD4+ or CD8+ T cells, the parasitized cells in the lymph nodes on day 11 of infection were all within the CD4+ and CD8+ populations, respectively, indicating that there had been minimal transfer of the parasite between cell types. Phenotypic analyses of cultures of PBMC infected in vitro with saturating concentrations of sporozoites revealed that parasitized B cells were abundant in the cultures after 1 week but were subsequently overgrown by T cells. The results of these experiments indicate that the cell type infected by T. parva influences the pathogenicity of the parasite.

Adhesion of flowing leucocytes to immobilized platelets.

Adhesion of neutrophils, lymphocytes and promyelocytic HL60 cells was compared in a flow-based model in which a monolayer of activated platelets formed the adhesive substrate. Each type of leucocyte formed P-selectin-mediated rolling attachments on the platelet surface under physiologically relevant flow conditions. Lymphocytes adhered less, and HL60 in similar numbers, compared to neutrophils, whereas the lymphocytes and HL60 cells rolled much more rapidly. Sulphated, sialylated saccharide(s) were implicated as ligand(s) for P-selectin for all leucocytes, but L-selectin (borne by neutrophils and lymphocytes, but not HL60 cells) appears to be a major presenter of ligand for neutrophils alone. T cells enriched from peripheral blood lymphocytes adhered in greater numbers than B cells. Differentiation of HL60 cells to neutrophil-like cells (induced by DMSO) caused cell volume to decrease and surface expression of integrin adhesion molecules to increase, but only a small percentage of cells were converted to an L-selectin-bearing phenotype. Differentiated cells showed evidence of stabilization of adhesion with increasing stress and a marked reduction in rolling velocity. These studies indicate that cell differentiation may be accompanied by alteration of adhesive behaviour, resulting from changes in physical characteristics as well as surface properties. Moreover, results suggest that P-selectin could promote lymphocyte attachment to endothelium in acute inflammatory conditions and possibly mediate lymphocyte-platelet interaction during thrombosis.

Antigen-driven B cell differentiation in vivo.

The secretion of specific antibodies and the development of somatically mutated memory B cells in germinal centers are consequences of T cell-dependent challenge with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). Using six-parameter flow cytometry and single cell molecular analysis we can directly monitor the extent of somatic hypermutation in individual responsive (isotype switched) antigen-specific B cells. The current study provides a direct quantitative assessment of recruitment into the antibody-secreting compartment on the one hand, and the germinal center pathway to memory on the other. Cellular expansion in both compartments is exponential and independent during the first week after challenge. The first evidence of somatic mutation, towards the end of the first week, was restricted to the germinal center pathway. Furthermore, germinal center cells express a significantly shorter third hypervariable region (CDR3), even when unmutated, than their antibody-secreting counterparts, suggesting a secondary selection event may occur at the bifurcation of these two pathways in vivo. By the end of the second week, the majority of mutated clones express a shorter CDR3 and affinity-increasing mutations as evidence of further selection after somatic mutation. These data provide evidence for substantial proliferation within germinal centers before the initiation of somatic mutation and the subsequent selection of a significant frequency of mutated clonotypes into the memory compartment.