Validation of a new in vitro Sun Protection Factor method to include a wide range of sunscreen product emulsion types.

In 2017, Cosmetics Europe performed a double-blinded ring test of 24 emulsion-type sunscreen products, across 3 in vivo test laboratories and 3 in vitro test laboratories, using a new candidate in vitro SPF test method. Based on the results of this work, an article was published showing how data derived from a new lead candidate method conform to new International Standards (ISO) acceptance criteria for alternative SPF test methods (Any alternative method should consider the matrix effect and if required, specify the matrix applicability of the method; Criterion 1a: Systematic differences between methods should be negligible: 95% of all individual results of an alternative method are within the range of ±2× reproducibility standard deviation of the in vivo method, that is overall bias must be below 0.5× reproducibility standard deviation of the in vivo method; Criterion 1b: Measurement uncertainty of an alternative method should be below the measurement uncertainty of the in vivo method. Candidate method predicted values must fall within the full 'funnel' (SPF 6-50+) limits proposed by Cosmetics Europe (derived from the same minimum test design, that is using the ISO24444 Method to measure at least 24 products across at least 3 laboratories using at least 5 test subjects/laboratory, in a blinded fashion).). Of the 24 sunscreen products tested, the majority of emulsions were of the oil-in-water (O/W) type, whereas only one was water-in-oil (W/O) and there were no products with a mineral-only sun filter system. In order to confirm the scope of this method, therefore, a new study was conducted that included 73 W/O (12 mineral + organic, 44 mineral only and 17 organic only) and 3 O/W mineral-only, emulsion-type sunscreen products (a total of 76 new sunscreen products). When combined with the previous 24 products (tested in 3 different laboratories), this yielded a new data set comprising a total of 100 emulsion-type sunscreen products, with SPF values ranging from 6 to 50+ (with a total of 148 data points). These products were tested using the double-plate in vitro SPF test method and compared with the ISO TC217/WG7 acceptance criteria for alternative SPF test methods. Over 95% of paired in vitro: in vivo SPF values lay within the upper and lower limits of the ISO acceptance criteria funnel, with no bias. This new in vitro SPF test method, therefore, meets the minimum requirements for an alternative SPF test method to ISO24444:2010, for emulsion-type sunscreen products (which make up the majority of marketed sunscreen products).

En 2017, Cosmetics Europe a réalisé un ring test en double aveugle de 24 produits de protection solaire de type émulsion, dans 3 laboratoires de test in vivo et 3 laboratoires de test in vitro, en utilisant une nouvelle méthode de test SPF in vitro. Sur la base des résultats de ces travaux, un article a été publié montrant comment les données dérivées de cette nouvelle méthode sont conformes aux nouveaux critères d'acceptation des normes internationales (ISO) pour les méthodes de test SPF alternatives. Sur les 24 produits de protection solaire testés, la majorité des émulsions étaient du type huile dans l'eau (H / E), tandis qu'un seul était de l'eau dans l'huile (E / H) et il n'y avait aucun produit contenant uniquement des minéraux. Afin de confirmer cette méthode, une nouvelle étude a donc été menée comprenant 73 produits E/ H (12 produits contenant des filtres minéraux + organiques, 44 produits contenant des filtres minéraux uniquement et 17 produits contenant des filtres organiques uniquement) et 3 produits H / E contenant des filtres minéraux uniquement, tous de type émulsion (donc un un total de 76 nouveaux produits de protection solaire). Combiné aux 24 produits précédents (testés dans 3 laboratoires différents), cela a donné un nouvel ensemble de données comprenant un total de 100 produits de protection solaire de type émulsion, avec des valeurs SPF allant de 6 à 50+ (avec un total de 148 points de données) . Ces produits ont été testés à l'aide de la méthode de test SPF in vitro double approche et comparés aux critères d'acceptation de l'ISO TC217 / WG7 pour les méthodes alternatives du SPF in vivo. Plus de 95% des valeurs de SPF appariées in vitro: in vivo se situent dans les limites supérieure et inférieure de l'entonnoir des critères d'acceptation ISO, sans biais. Cette nouvelle méthode de test SPF in vitro, par conséquent, répond aux exigences minimales d'une méthode de test SPF alternative à ISO24444: 2010, pour les produits de protection solaire de type émulsion (qui constituent la majorité des produits de protection solaire commercialisés).

Anti-inflammatory / anti-oxidant activity of ingredients of sunscreen products? Implications for SPF.

OBJECTIVE: The Sun Protection Factor (SPF) of sunscreen products is derived from testing in vivo their ability to prevent erythema ("sunburn"). Recently, certain articles have raised concerns that sunscreen products may actively suppress erythema via anti-inflammatory / anti-oxidant (AI/AO) activity. These articles reason that this may result in a higher labelled SPF value than that provided by the efficacy of the UVR filters alone, giving consumers a "false sense of security". On the other hand, since inflammatory processes are known to play a role in the mechanisms of photodamage / skin cancer induction and propagation, AI/AO activity may provide valuable incremental photoprotective benefit (provided that there is no interference with visible erythema). The objective of these studies, therefore, was to investigate the potential of AI/AO ingredients to suppress UVR-induced erythemal response in human skin, in vivo. METHODS: In vivo studies with SPF30 sunscreen formulations containing a variety of AI/AO ingredients were performed according to the International Standard ISO24444:2010 method. While ISO24444:2010 requires assessment of erythema at 20 ± 4h post-irradiation, an additional assessment at 5 h post-irradiation was also used to determine potential delay in erythema development. RESULTS: None of the formulations, containing a variety of AI/AO ingredients, influenced SPF determination in comparison to the vehicle formulation. CONCLUSION: Our in vivo results demonstrate that commonly-used AI/AO ingredients, at concentrations typically used in sunscreen products, neither influence SPF value nor delay erythemal response, i.e., the measured SPF reflects the true photoprotective capacity of the product.

OBJECTIF: Le facteur de protection solaire (SPF) des produits de protection solaire est dérivé de tests in vivo servant à déterminer leur capacité à prévenir un érythème (« coup de soleil ¼). Récemment, certains articles ont soulevé des inquiétudes en insinuant que les produits de protection solaire pourraient activement faire disparaître un érythème par le biais d'une activité anti-inflammatoire/anti-oxydante (AI/AO). Ces articles soutiennent que cela pourrait impliquer une valeur déclarée du SPF plus élevée que celle fournie par l'efficacité des filtres RUV à eux seuls, donnant ainsi une « fausse impression de sécurité ¼ aux consommateurs. D'autre part, étant donné que les processus inflammatoires sont réputés jouer un rôle dans les mécanismes de photo-altération/d'induction et de propagation du cancer de la peau, l'activité AI/AO pourrait apporter un précieux bénéfice photo-protecteur amplifié (à condition qu'il n'y ait aucune interférence avec un érythème visible). L'objectif de ces études était, par conséquent, d'étudier le potentiel des ingrédients contribuant à l'activité AI/AO à faire disparaître la réponse érythémateuse induite par les RUV dans la peau humaine, in vivo. MÉTHODES: Des études in vivo avec des formules de produits solaires à SPF30 contenant une variété d'ingrédients contribuant à l'activité AI/AO ont été effectuées conformément à la méthode correspondant à la norme internationale ISO24444:2010. Bien que l'ISO24444:2010 nécessite l'évaluation de l'érythème à 20 _ 4 heures post-irradiation, une évaluation supplémentaire à 5 heures post-irradiation a également été utilisée pour déterminer l'éventuel délai d'apparition d'un érythème. RÉSULTATS: Aucune des formules, contenant une variété d'ingrédients contribuant à l'activité AI/AO, n'a influencé la détermination du SPF par comparaison à la formule véhicule. CONCLUSION: Nos résultats in vivo démontrent que les ingrédients contribuant à l'activité AI/AO fréquemment utilisés, aux concentrations généralement utilisées dans les produits de protection solaire, n'influencent pas la valeur du SPF, pas plus qu'ils ne retardent la réponse érythémateuse, autrement dit, le SPF mesuré reflète la véritable capacité photo-protectrice du produit.

Validation of an in vitro sun protection factor (SPF) method in blinded ring-testing.

OBJECTIVE: The objective of this work was to investigate the utility of a new in vitro SPF test method in blinded ring-testing, against new ISO acceptance criteria. METHODS: Twenty four blinded, commercial, emulsion-type, primary sunscreen products, covering the full range of labelled SPF in Europe (SPF6 - 50+), were tested by three test institutes using the current ISO24444:2010 In Vivo SPF Test Method and simultaneously by three separate test laboratories using a new candidate in vitro SPF test method, developed under the leadership of Cosmetics Europe (CE). The resulting relationship between in vitro SPF and in vivo SPF values was then compared with acceptance criteria developed recently by the International Standards (ISO) TC217/WG7 Sun Protection Test Methods Working Group. RESULTS: Analysis of the mean inter-laboratory in vitro and mean inter-institute in vivo SPF values revealed a strong correlation between in vitro and in vivo values, with a Pearson correlation coefficient of r2  = 0.88 (P < 0.0001), a slope of 1.01 and a non-significant intercept (-1.48; P = 0.62). When these data were compared to the new ISO WG7 acceptance criteria, method bias was found to be extremely low and over 95% of the coupled data lay within the model 'funnel' (defined by upper and lower confidence intervals). CONCLUSION: In conclusion, the results of blinded ring testing and comparison to new ISO WG7 acceptance criteria indicate that a new in vitro SPF test method meets (and exceeds) these minimum criteria and is an interesting candidate for possible deployment as an industry test methodology.

[Analysis of psychological factors influencing peritoneal dialysis selection]. / Análisis de la influencia de los factores psicológicos en la elección de diálisis peritoneal.

BACKGROUND: Peritoneal dialysis (PD) is not frequently used in our setting. OBJECTIVE: To analyze the psychological factors involved in the choice of renal replacement therapy (RRT). MATERIAL AND METHODS: A prospective observational study of stable patients without cognitive or sensory deficits who were informed about RRT from January 2004 to July 2006 and agreed to participate. The patients were given and completed the Beck Depression Inventory and the Eysenck personality questionnaire. Clinical and sociodemographic data and RRT choice were recorded. End of follow-up: 2007/10/31. RESULTS: 44 patients were studied: age, 65.4 +/- 13.1 years, 48% male, 34% diabetic. When choosing RRT, 36% of patients had symptoms of depression. Neither depression symptoms nor personality traits were related to the choice of dialysis type. The youngest patients chose PD (41%). After a mean followup of 8 +/- 8 months, 70% of patients started RRT (68% haemodialysis [HD], 32% PD). None of the patients who chose HD changed their mind, but 3 of the 13 patients (23%) who chose PD finally commenced HD, usually in the context of a worsening of the disease. Half of the patients with depression symptoms when choosing PD and a third of the patients with higher levels of neuroticism changed their decision and finally opted for HD. CONCLUSIONS: When choosing RRT, the prevalence of depression symptoms is high. Neither depression nor personality traits influenced the initial choice of RRT, although these factors may be involved in subsequent changes to the decision.

Divalent cation regulation of the function of the leukocyte integrin LFA-1.

The integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells serves as a useful model for analysis of leukocyte integrin functional activity. We have assessed the role of divalent cations Mg2+, Ca2+, and Mn2+ in LFA-1 binding to ligand intercellular adhesion molecule-1 (ICAM-1) and induction of the divalent cation-dependent epitope recognized by mAb 24. Manganese strongly promoted both expression of the 24 epitope and T cell binding to ICAM-1 via LFA-1, suggesting that Mn2+ is able to directly alter the conformation of LFA-1 in a manner that favors ligand binding. Since Mn2+ also promotes functional activity of other integrins, parallels in mechanism of ligand binding may span the integrin family. In contrast, induction of 24 epitope expression by Mg2+ required removal of Ca2+ from T cell LFA-1 with EGTA. Furthermore, binding of mAb 24 to T cell LFA-1 in the presence of either Mn2+ or Mg2+ was found to be specifically inhibited by Ca2+, suggestive of a negative regulatory role for Ca2+ in the control of leukocyte integrin function. Analysis of T cell binding to ICAM-1 via LFA-1 in the presence of Mg2+ or Mn2+, confirmed that Ca2+ exerted inhibitory effects upon LFA-1 function. The implication of our findings is that Ca2+ bound with relatively high affinity to LFA-1 may serve to maintain an inactive state. Thus induction of function and 24 epitope expression may occur as a result of displacement of Ca2+ from leukocyte integrins or alternatively, such activators may be able to impose the required conformational change in the presence of bound Ca2+.

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function.

The leukocyte integrins (CD11/CD18 or beta 2-type integrins) are expressed exclusively on leukocytes and participate in many adhesion-dependent functions (Arnaout, M.A. 1990. Blood. 75:1037-1050; Springer, T. A. 1990. Nature. (Lond.) 346:425-434; Dustin, M. L., and T. S. Springer. 1991. Annu. Rev. Immunol. 9:27-66). The avidity of leukocyte integrin binding to their ligands or counter-receptors is dependent upon response to intracellular signals (Wright, S. D., and B. C. Meyer. 1986. J. Immunol. 136:1759-1764; Dustin, M. A., and T. S. Springer. 1989. Nature (Lond.). 341:619-624). We have investigated the effects of a novel mAb (mAb 24) which defines a leukocyte integrin alpha subunit epitope that is Mg(2+)-dependent and may be used as a "reporter" of the activation state of these receptors (Dransfield, I., and N. Hogg. 1989. EMBO (Eur. Mol. Biol. Organ) J. 8:3759-3765; Dransfield, I., A.-M. Buckle, and N. Hogg. 1990. Immunol. Rev. 114:29-44; Dransfield, I., C. Cabañas, A. Craig, and N. Hogg. 1992. J. Cell Biol.) Data is presented to show that this mAb inhibits monocyte-dependent, antigen-specific T cell proliferation and IL-2-activated natural killer cell assays which are both dependent on lymphocyte function-associated antigen-1 (LFA-1), and complement receptor type 3 (CR3)-mediated neutrophil chemotaxis to f-Met-Leu-Phe. This inhibitory effect is not caused by the prevention of receptor/ligand binding because LFA-1/ICAM-1, LFA-1/ICAM-2,3 and CR3/iC3b interactions are, under activating conditions, promoted rather than blocked by mAb 24. As it does not interfere with mitogen-stimulated T cell proliferation, it is unlikely that mAb 24 transduces a "negative" or antiproliferative signal to the T cells to which it is bound. Using a model system of transient activation of LFA-1, we have found that mAb 24 prevents "deadhesion" of receptor/ligand pairs, possibly locking leukocyte integrins in an "active" conformation. It is speculated that inhibition of leukocyte integrin function by this mAb reflects the necessity for dynamic leukocyte integrin/ligand interactions.

ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes.

The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

The tyrosine kinase PYK-2/RAFTK regulates natural killer (NK) cell cytotoxic response, and is translocated and activated upon specific target cell recognition and killing.

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK-target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK-target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

The interaction of activated integrin lymphocyte function-associated antigen 1 with ligand intercellular adhesion molecule 1 induces activation and redistribution of focal adhesion kinase and proline-rich tyrosine kinase 2 in T lymphocytes.

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the beta2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the beta2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

Camptothecin induces differentiation and stimulates the expression of differentiation-related genes in U-937 human promonocytic leukemia cells.

We have studied the effect of the DNA topoisomerase I inhibitor camptothecin on growth, differentiation, and gene expression in U-937 human promonocytic leukemia cells. At a concentration of 20 nM, camptothecin caused significant DNA strand breakage and decreased the growth activity by accumulating cells preferentially at the G2 phase of the cycle. The growth arrest occurred concomitantly with an increase in cell size. Under those conditions, camptothecin induced differentiation, as demonstrated by (a) the capacity of the cells to generate reactive oxygen species, (b) the increase in the surface expression of the leukocyte integrins CD11b/CD18 and CD11c/CD18, (c) the increase in the cellular content of the intermediate filament protein vimentin, and (d) the decrease in the surface expression of the transferrin receptor. Camptothecin also induced the expression of differentiation markers in other human myeloid cells, namely, the promonocytic THP-1 and the myelomonocytic HL-60 cell lines. Northern blot assays revealed that camptothecin stimulated the expression of CD11b, CD11c, and vimentin at the mRNA level. Moreover, the drug increased the transcription rate of the vimentin gene, as shown by "run-on" transcription assays.

Induction of LFA-1-mediated homotypic adhesions in promonocytic U-937 cells occurs independently of cell differentiation.

The differentiation of monocytes into macrophages occurs along with a marked increase in LFA-1-dependent intercellular adhesions. Similarly, the phorbol ester-induced differentiation of U-937 promonocytic cells into macrophage-like cells is morphologically characterized by an important increase in LFA-1/ICAM-1-dependent intercellular homotypic adhesions. Since an important functional role in activation of human T cells has been demonstrated for LFA-1-dependent adherence, we have analyzed whether the induction of LFA-1-dependent intercellular adhesion of human monocytic cells is necessarily accompanied by differentiation of these cells. We found that treatment of the promonocytic U-937 cells with the anti-LFA-1 mAb NKI-L16 induces formation of intercellular clusters, but does not induce cell differentiation as determined by several differentiation markers. These markers include the arrest of cell proliferation, production of reactive oxygen species, changes in the cell surface expression of differentiation-associated antigens such as the transferrin receptor, CD11b and CD11c and changes in the levels of several specific gene transcripts such as CD18 antigen, c-myc, ornithine decarboxylase and vimentin. These findings suggest that LFA-1-dependent adhesion and differentiation of monocytic cells are independent processes.

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.

Differential expression of activation epitopes of beta1 integrins in psoriasis and normal skin.

In the epidermis, beta1 integrin expression is normally confined to the basal layer; however, suprabasal expression of beta1 integrins in keratinocytes has been found in psoriasis, and it has been suggested that it could be a pathogenic factor of the disease. We have investigated herein the functional state of beta1 integrins of human keratinocytes in normal skin and psoriasis. The expression of beta1-activation-reporter epitopes was monitored with two monoclonal antibodies, HUTS-21 and MG5A7, that recognize epitopes whose expression parallels functional activity of beta1 integrins and correlates with the ligand binding activity of these heterodimeric glycoproteins. We have found that keratinocytes express activation epitopes of beta1 integrins, and that these epitopes can be modulated by manganese. The expression of activation epitopes of beta1 integrins was related to an enhanced adhesion to fibronectin and collagen. Immunohistochemical studies of normal and psoriatic skin with HUTS-21 and other monoclonal antibodies indicate that, although there is suprabasal expression of beta1 integrins in psoriasis, these molecules seem to be in an inactive state. Moreover, most beta1 integrins in lateral and apical surfaces of basal keratinocytes of psoriasis are also in a nonactive conformation, implying a decrease of activity compared with normal skin, in which active beta1 integrins are distributed all over the basal keratinocytes.

Lymphocyte-fibroblast adhesion. A useful model for analysis of the interaction of the leucocyte integrin LFA-1 with ICAM-1.

The adhesion of human T lymphoblasts to ICAM-1-expressing normal dermal fibroblasts has been assessed as a sensitive model system for the analysis of the interaction of the leucocyte integrin LFA-1 with its counter-receptor ICAM-1. Using this model system, the effects of factors known to regulate the activity of LFA-1 have been quantitated: temperature; concentration of divalent cations; and exposure to phorbol esters. We show here that under the appropriate assay conditions, this model system represents a useful and simple alternative to the detection of leucocyte binding to purified ICAM-1 and also has the additional advantage of permitting more sensitive quantification than is possible using the homotypic adhesion assay.

Functional regulation of the human integrin VLA-1 (CD49a/CD29) by divalent cations and stimulatory beta 1 antibodies.

We have investigated the regulation by divalent cations Mg2+, Ca2+ and Mn2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg2+ or microM levels of Mn2+. In contrast, Ca2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg(2+)-supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin beta 1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-alpha 1 specific monoclonal antibody, 5E8D9.

Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma.

Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

S-phase inhibitors induce vimentin expression in human promonocytic U-937 cells.

The administration of hydroxyurea (3 x 10(-4) M) and cytosine arabinoside (10(-7) M) greatly induces the expression of the vimentin gene in human promonocytic leukemia U-937 cells. The induction takes place at both the mRNA and protein levels, as demonstrated by Northern blot, immunoblot and immunofluorescence assays. On the contrary, the drugs inhibit the expression of c-myc and ornithine decarboxylase, and do not modify significantly the expression of beta-actin. Since hydroxyurea and cytosine arabinoside trigger the phenotypic differentiation of U-937 cells, as demonstrated by the induction of the differentiation-specific CD11b and CD11c antigens, it is concluded that vimentin expression might be implicated in the maturation of these cells.

Endoglin is expressed in the chicken vasculature and is involved in angiogenesis.

Endoglin is a component of the transforming growth factor beta (TGF-beta) receptor complex, highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant vascular disorder caused by a haploinsufficiency mechanism. Vascular lesions (telangiectasia and arteriovenous malformations) in HHT1 are associated with loss of the capillary network, suggesting the involvement of endoglin in vascular repair processes. Using the chick chorioallantoic membrane (CAM) as an angiogenic model, we have analyzed the expression and function of chicken endoglin. A pan-specific polyclonal antibody (pAb) recognized chicken endoglin as demonstrated by immunostaining and Western blot analysis. In ovo treatment of chicken embryos with this pAb resulted in a significantly increased area of CAM. This effect was likely mediated by modulation of the ligand binding to endoglin as this pAb was able to inhibit TGF-beta1 binding. These results support the involvement of endoglin in the angiogenic process.