Neosaxitoxin Inhibits the Expression of Inflammation Markers of the M1 Phenotype in Macrophages.

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 µM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1ß, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

Anthracycline-related cardiotoxicity in patients with breast cancer harboring mutational signature of homologous recombination deficiency (HRD).

BACKGROUND: The BRCA proteins play a key role in the homologous recombination (HR) pathway. Beyond BRCA1/2, other genes are involved in the HR repair (HRR). Due to the prominent role in the cellular repair process, pathogenic or likely pathogenic variants (PV/LPVs) in HRR genes may cause inadequate DNA damage repair in cardiomyocytes. PATIENTS AND METHODS: This was a multicenter, hospital-based, retrospective cohort study to investigate the heart toxicity from anthracycline-containing regimens (ACRs) in the adjuvant setting of breast cancer (BC) patients carrying germline BRCA PV/LPVs and no-BRCA HRR pathway genes. The left ventricular ejection fraction (LVEF) was assessed using cardiac ultrasound before starting ACR therapy and at subsequent time points according to clinical indications. RESULTS: Five hundred and three BC patients were included in the study. We predefined three groups: (i) BRCA cohort; (ii) no-BRCA cohort; (iii) variant of uncertain significance (VUS)/wild-type (WT) cohort. When baseline (T0) and post-ACR (T1) LVEFs between the three cohorts were compared, pre-treatment LVEF values were not different (BRCA1/2 versus HRR-no-BRCA versus VUS/WT cohort). Notably, during monitoring (T1, median 3.4 months), patients carrying BRCA or HRR no-BRCA germline pathogenic or likely pathogenic variants showed a statistically significant reduction of LVEF compared to baseline (T0). To assess the relevance of HRR on the results, we included the analysis of the subgroup of 20 BC patients carrying PV/LPVs in other genes not involved in HRR, such as mismatch repair genes (MUTYH, PMS2, MSH6). Unlike HRR genes, no significant differences in T0-T1 were found in this subgroup of patients. CONCLUSION: Our data suggest that deleterious variants in HRR genes, leading to impaired HR, could increase the sensitivity of cardiomyocytes to ACR in early BC patients. In this subgroup of patients, other measurements, such as the global longitudinal strain, and a more in-depth assessment of risk factors may be proposed in the future to optimize cardiovascular risk management and improve long-term survival.

Notch signalling regulates cytokine production by CD8+ and CD4+ T cells.

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.

Differential regulation of Notch ligands in dendritic cells upon interaction with T helper cells.

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.

A large view of CYP21 locus among Sicilians and other populations: identification of a novel CYP21A2 variant in Sicily.

BACKGROUND: Several mutations in CYP21 locus cause 21-hydroxylase deficiency (21-OHD). The most common mutations are widespread among different geographic areas and their frequencies have been also reported to differ among certain populations. AIM: To obtain a large view on the frequencies of the most common mutations in the CYP21 locus, in Sicily, in the Mediterranean and other major geographic areas worldwide. SUBJECTS AND METHODS: Three hundred and eight unrelated CYP21A2 alleles leading 21-OHD in Sicily were genetically typed and compared with other series previously reported in Sicily and in surrounding regions. An analysis of the frequencies of the different geographic areas was also carried out. CYP21A2 typing was carried out using PCR-restriction fragment length polymorphism (RFLP), for the detection of the CYP21A2 deletion, while sequencing analysis was performed to evaluate all the missense/non-sense mutations. RESULTS: Our study revealed that p.V281L (44.4%), I2splice (21.6%) and p.P30L (11.2%) were very frequent alleles, del8bp (0.4%) was found very rarely in Sicily and a novel mutation leading to non-classical phenotype, p.L198F, was also discovered in this population. Allele frequencies were found to be significantly different from previously observed frequencies in Sicily. In addition, here we present the most significant frequency modifications among different geographic areas worldwide. CONCLUSIONS: As the distribution of the disease CYP21A2 alleles is heterogeneous around the world, the knowledge of the relative distributions allows a better management of 21-OHD for fetuses and newborns in different geographic areas.

Impact of deleterious variants in other genes beyond BRCA1/2 detected in breast/ovarian and pancreatic cancer patients by NGS-based multi-gene panel testing: looking over the hedge.

BACKGROUND: Hereditary breast cancer (BC), ovarian cancer (OC), and pancreatic cancer (PC) are the major BRCA-associated tumours. However, some BRCA1/2-wild-type (wt) patients with a strong personal and/or family history of cancer need a further genetic testing through a multi-gene panel containing other high- and moderate-risk susceptibility genes. PATIENTS AND METHODS: Our study was aimed to assess if some BC, OC, or PC patients should be offered multi-gene panel testing, based on well-defined criteria concerning their personal and/or family history of cancer, such as earliness of cancer onset, occurrence of multiple tumours, or presence of at least two or more affected first-degree relatives. For this purpose, 205 out of 915 BC, OC, or PC patients, resulted negative for BRCA1/2 and with significant personal and/or family history of cancer, were genetically tested for germline pathogenic or likely pathogenic variants (PVs/LPVs) in genes different from BRCA1/2. RESULTS: Our investigation revealed that 31 (15.1%) out of 205 patients harboured germline PVs/LPVs in no-BRCA genes, including PALB2, CHEK2, ATM, MUTYH, MSH2, and RAD51C. Interestingly, in the absence of an analysis conducted through multi-gene panel, a considerable percentage (15.1%) of PVs/LPVs would have been lost. CONCLUSIONS: Providing a multi-gene panel testing to BRCA1/2-wt BC/OC/PC patients with a strong personal and/or family history of cancer could significantly increase the detection rates of germline PVs/LPVs in other cancer predisposition genes beyond BRCA1/2. The use of a multi-gene panel testing could improve the inherited cancer risk estimation and clinical management of patients and unaffected family members.

Correction to: Order versus chaos: The impact of structure on number-space associations.

In the Results section (pp.1785, left column), please replace the sentences "In particular, RTs for the large-unstructured condition were significantly faster than RTs on the small-unstructured condition.

Hohlraum-driven mid-Z (SiO2) double-shell implosions on the omega laser facility and their scaling to NIF.

High-convergence, hohlraum-driven implosions of double-shell capsules using mid-Z (SiO2) inner shells have been performed on the OMEGA laser facility [T. R. Boehly, Opt. Commun. 133, 495 (1997)]. These experiments provide an essential extension of the results of previous low-Z (CH) double-shell implosions [P. A. Amendt, Phys. Rev. Lett. 94, 065004 (2005)] to materials of higher density and atomic number. Analytic modeling, supported by highly resolved 2D numerical simulations, is used to account for the yield degradation due to interfacial atomic mixing. This extended experimental database from OMEGA enables a validation of the mix model, and provides a means for quantitatively assessing the prospects for high-Z double-shell implosions on the National Ignition Facility [Paisner, Laser Focus World 30, 75 (1994)].

Order versus chaos: The impact of structure on number-space associations.

The Spatial-Numerical Association of Response Codes (SNARC) effect has been observed with different stimuli, beside Arabic numerals, such as written/spoken number words, sequences of acoustic stimuli, and groups of elements. Here we investigated how the enumeration of sets of elements can be affected by the spatial configuration of the displayed stimuli with regard to the emergence of the SNARC effect. To this aim, we asked participants to perform a magnitude comparison task with structured (i.e., dice-like) and unstructured (i.e., random) patterns of rectangles. With this manipulation, we sought to explore the presence of the SNARC effect in relation to the structure of the displayed visual stimuli. The results showed that the spatial arrangement of rectangles does not impact visual enumeration processes leading to the SNARC effect. An unexpected reversal of the size effect for unstructured stimuli was also observed. We speculate that the presence of a similar SNARC effect, both with structured and unstructured stimuli, indicates the existence of a common access to the mental number line.

Alemtuzumab induction in kidney transplantation: clinical results and impact on T-regulatory cells.

Alemtuzumab (ALT), a humanized monoclonal anti-CD52 antibody, was introduced in solid organ transplantation as an induction agent. ALT associated with anticalcineurins has provided a low incidence of acute rejection episodes (ARE) and potential tolerogenic properties. We analyzed the clinical outcomes and effects on peripheral Treg of renal transplant recipients treated with ALT. Six-month data on kidney alone or kidney combined with pancreas or liver patients treated with ALT and tacrolimus (TAC) in standard doses were compared with those on renal transplant recipients of similar demography who were not treated with ALT. We evaluated patient and graft survivals, ARE incidence, hematological parameters, renal function, adverse events, and CD4+CD25+FoxP3+ T cells in peripheral blood. Demographics of recipients, donors, and transplants were similar in both groups. Mean HLA mismatch was slightly greater among ALT-treated patients (3.5 vs 2.5). No combined transplantation was performed in the ALT-untreated group. Patient and graft survivals were 100% without rejection or serious infections in both groups. ALT-treated recipients showed anemia and leukopenia in 3 patients as well as severe lymphopenia in 5 recipients, who partially recovered on day 90. Final mean plasma creatinine was 1.4 mg/dL, while calculated creatinine clearance was approximately 65 mL/min in both groups. Mean Treg cell percentage was higher among ALT-treated recipients than the comparative group or healthy controls (P < .05). In conclusion, renal transplantation results obtained using ALT with rigorous immunosuppressive therapy were excellent; serious adverse events and acute rejection were absent. The effect of the increased proportion of Treg cells must be evaluated with longer observation.

Generation of dendritic cells with regulatory properties.

Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D(3) and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D(3) generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.

A machine learning tool for re-planning and adaptive RT: A multicenter cohort investigation.

PURPOSE: To predict patients who would benefit from adaptive radiotherapy (ART) and re-planning intervention based on machine learning from anatomical and dosimetric variations in a retrospective dataset. MATERIALS AND METHODS: 90 patients (pts) treated for head-neck cancer (H&N) formed a multicenter data-set. 41 H&N pts (45.6%) were considered for learning; 49 pts (54.4%) were used to test the tool. A homemade machine-learning classifier was developed to analyze volume and dose variations of parotid glands (PG). Using deformable image registration (DIR) and GPU, patients' conditions were analyzed automatically. Support Vector Machines (SVM) was used for time-series evaluation. "Inadequate" class identified patients that might benefit from replanning. Double-blind evaluation by two radiation oncologists (ROs) was carried out to validate day/week selected for re-planning by the classifier. RESULTS: The cohort was affected by PG mean reduction of 23.7±8.8%. During the first 3weeks, 86.7% cases show PG deformation aligned with predefined tolerance, thus not requiring re-planning. From 4th week, an increased number of pts would potentially benefit from re-planning: a mean of 58% of cases, with an inter-center variability of 8.3%, showed "inadequate" conditions. 11% of cases showed "bias" due to DIR and script failure; 6% showed "warning" output due to potential positioning issues. Comparing re-planning suggested by tool with recommended by ROs, the 4th week seems the most favorable time in 70% cases. CONCLUSIONS: SVM and decision-making tool was applied to overcome ART challenges. Pts would benefit from ART and ideal time for re-planning intervention was identified in this retrospective analysis.

Evolution of sex determination in caenorhabditis: unusually high divergence of tra-1 and its functional consequences.

The tra-1 gene is a terminal regulator of somatic sex in Caenorhabditis elegans: high tra-1 activity elicits female development, low tra-1 activity elicits male development. To investigate the function and evolution of tra-1, we examined the tra-1 gene from the closely related nematode C. briggsae. Ce-tra-1 and Cb-tra-1 are unusually divergent. Each gene generates two transcripts, but only one of these is present in both species. This common transcript encodes TRA-1A, which shows only 44% amino acid identity between the species, a figure much lower than that for previously compared genes. A Cb-tra-1 transgene rescues many tissues of tra-1(null) mutants of C. elegans but not the somatic gonad or germ line. This transgene also causes nongonadal feminization of XO animals, indicating incorrect sexual regulation. Alignment of Ce-TRA-1A and Cb-TRA-1A defines several conserved regions likely to be important for tra-1 function. The phenotypic differences between Ce-tra-1(null) mutants rescued by Cb-tra-1 transgenes and wild-type C. elegans indicate significant divergence of regulatory regions. These molecular and functional studies suggest that evolution of sex determination in nematodes is rapid and genetically complex.

Pre-dialysis application of the glucose infusion test for recirculation detection.

Vascular access recirculation (R) is a well-known cause of decreased dialysis dose. In this paper a new easy protocol for R detection in pre-dialysis derived from the classic Glucose Infusion Test (GIT) is introduced. The pre-dialysis GIT (GIT-pre) is based on the glucose (5%, 10 ml) bolus injection directly into the venous needle and on a simultaneous withdrawal from the arterial needle. If the glucose value increases during the glucose bolus, R is present. This new protocol was validated on 29 chronic haemodialysis patients (20 AVFs, 7 CVCs, 2 PTFE grafts), comparing the glucose increase with the classic GIT during dialysis. Only one CVC had R with the blood lines in the normal position (deltaglu = 465 mg/dl with GIT-pre and a deltaglu = 186 mg/dl, R = 9.3% with classic GIT) while in the reverse blood line position, all CVCs showed a significant glucose increase (mean GIT-pre deltaglu = 195 mg/dl; mean GIT deltaglu = 140 mg/dl corresponding to a R = 8%). There were 5 AVFs with true R (correct blood lines position) clearly identified by both methods (mean values deltaglu = 316 mg/dl with GIT-pre and a deltaglu = 390 mg/dl, R = 19.5% with classic GIT). Preliminary results show good reliability of the new protocol in identifying VA R caused either by failing VA with stenosis or by reverse blood lines position. The GIT-pre is a simpler application of the classic GIT useful for testing new VA, new needle positions or CVC performance before starting dialysis. A simpler R test could increase the frequency of the measurements and consequently the power of R in early detection of VA problems.

The human SRY protein is present in fetal and adult Sertoli cells and germ cells.

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.

Pro-Inflammatory Cytokines in Nasopharyngeal Aspirate From Hospitalized Children With Respiratory Syncytial Virus Infection With or Without Rhinovirus Bronchiolitis, and Use of the Cytokines as Predictors of Illness Severity.

Respiratory syncytial virus (RSV) and human rhinovirus (HRV) respiratory infection in children induce production of inflammatory interleukins (ILs) in the respiratory epithelium. As IL(s) determine the severity of illness, the purpose of this study was to identify the pro-inflammatory IL(s) that could be predictor(s) of clinical severity. One hundred and fifteen patients <2 years old with bronchiolitis due to RSV and /or HRV and 38 controls were selected from a hospital and an outpatient clinic. Clinical data of all patients were recorded. Severity was defined by the number of days with oxygen need. Nasopharyngeal aspirates (NPA) were collected to perform viral diagnosis by quantitative reverse transcription and polymerase chain reaction (qRT-PCR) and to quantify ILs: TNF-α, IL-10, IL-6, IL-1ß, and IL-8, by flow cytometry. Simple and multiple regression and receiver operating characteristic (ROC) curves were used for statistical analysis. Of the patients selected 60 were single RSV, 28 RSV associated to HRV, and 27 single HRV. All patients (115) showed significantly higher IL levels when compared with controls. Levels of IL-6, IL-1ß, and IL-8 detected in NPA from RSV single and associated to HRV were significantly higher than HRV infected and positively associated with days requiring O2.Levels of IL-6, IL-1ß, and IL-8 detected in NPA from patients infected with RSV only or with both RSV and HRV are increased, and any of those 3 cytokines may have a predictive value for the number of days with need of supplemental oxygen.

Lymphocyte adhesion to endothelium derived from human lymphoid tissue.

The development of an efficient immune response depends on the capacity of antigen-specific lymphocytes to migrate into secondary lymphoid organs. The first step in the process of lymphocyte extravasation involves lymphocyte binding to the vascular endothelium. Although several adhesion receptors have been implicated in the migration of lymphocytes to inflamed tissue, their role in the extravasation of these cells to normal lymphoid organs is not yet clearly established. The involvement of adhesion molecules in lymphocyte entrance to secondary lymphoid organs can be better assessed in an in vitro system using endothelial cells in culture. Here we report on the isolation and culture of a homogeneous population of adherent cells of endothelial origin derived from human tonsils (TEC) and on adhesion studies performed with these cells. Beginning from primary cultures of human tonsils, we isolated a population of cells that we show by FACScan analysis to present the intracellular endothelial cell marker Von Willebrand factor and LVAP-2, a surface molecule present in venules from lymphoid organs. The cells are negative for FDC, IDC and macrophage markers. They express ICAM-1, VCAM-1 and CD40 both constitutively and in inducible forms and are induced by IFN-gamma to express major histocompatibility complex class II antigens. As opposed to endothelial cells from human umbilical cord (HUVEC), they do not need to be activated by cytokines to bind lymphoid cells via VLA-4. The mAb HP2/1 directed to the integrin VLA-4 blocks adhesion of Ramos and Daudi cells to tumor necrosis factor alpha (TNF-alpha)-treated HUVEC and to untreated TEC but not of tonsil-derived MNC. On the other hand, an anti-VCAM-1 antibody that blocks adhesion of Ramos and Daudi cells to TNF-alpha-treated HUVEC, does not block adhesion of these cells to TEC, suggesting the presence on the tonsillar endothelial cells of a ligand for VLA-4 different from VCAM-1. We show here that this ligand is not fibronectin.

Spleen-derived stromal cells. Adhesion molecules expression and lymphocyte adhesion to reticular cells.

The final steps of lymphocyte differentiation occur in secondary lymphoid organs where B and T lymphocytes interact with the lymphoid microenvironment. Although numerous studies describe the interactions of murine lymphocytes with dendritic, follicular and other antigen presenting cells, little is known on the interactions between lymphocytes and reticular cells, an important cellular component of spleen stroma. In this work we describe the culturing of complete murine spleen stromas and of two cell lines, Sp-1 and Sp-2, identified as of possible reticular origin, and describe the adhesive interactions between murine lymphocytes and human lymphoid cells with murine spleen stromal cells. FACS analysis indicates that the Sp-1 cell line shows a single cell type expressing VCAM-1 and CD44 constitutively. They do not express any of the markers described for follicular cells, interdigitating cells, macrophages or endothelial cells. Our data suggests that these cells represent a population of spleen reticular cells. The Sp-2 cell line shows two phenotypically different cell types that grow in association. FACS analysis demonstrates that both cell types express VCAM-1 and CD44 constitutively, but that they can be differentiated by the expression of CD11b and FcR. These data suggest that the Sp-2 cell line is composed of one type of stromal cell growing over an adherent layer of reticular cells. Furthermore, analysis of the non-B non-T cell fraction prepared from murine spleen shows that approximately 30% of these cells correspond to the CD44/VCAM-1 double positive cells. Murine B and T cells adhere to the complete stromas and to Sp-1 and Sp-2 cell lines. Activation of B cells with LPS had no effect on binding while binding of T cells to complete stromas increased up to threefold after Con-A treatment. Adhesion of human lymphoblastoid Daudi cells to complete spleen stromas is blocked by an anti-(murine) VCAM-1 antibody but not by an antibody to the (human) integrin alpha 4 subunit, while adhesion to the Sp-1 and Sp-2 stromas is blocked by antibodies against both molecules. Also, adhesion of Ramos cells to Sp-2 stromas is inhibited by antibodies to the integrin alpha 4 subunit and to murine VCAM-1. Antibodies to other adhesion receptors such as the integrin beta 2 subunit, ICAM-1 or CD44 have no effect on human cell binding to these stromas. Our results suggest that we have isolated a fraction of splenic reticular cells and that these cells can be cultured as a distinct cell line. The finding that these cells express CD44 and VCAM-1 constitutively and use some of these molecules for lymphocyte binding suggests that spleen reticular cells may be involved in the regulation of normal lymphocyte traffic through the spleen.

Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity.

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA.