Community-acquired meticillin-resistant Staphylococcus aureus strain USA300 resists staphylococcal protein A modulation by antibiotics and antimicrobial peptides.

Community-acquired meticillin-resistant Staphylococcus aureus (CA-MRSA) causes severe diseases through virulence factors such as staphylococcal protein A (SpA), which favours immune evasion. We have previously shown that antimicrobial peptides (AMPs) and antibiotics decrease SpA expression in CA-MRSA strains. Here we examined the effects of antibiotics and AMPs, alone and in combination, on SpA expression in various CA-MRSA strains. Six S. aureus isolates corresponding to the major worldwide CA-MRSA clones (ST8-USA300, ST80 and ST30) were selected. Strains were cultured to exponential growth phase and were subsequently incubated with antibiotics (tigecycline, linezolid, clindamycin and vancomycin) at 0.25× MIC or with AMPs [human neutrophil peptide (HNP)-1-3] at the LD50, alone and in combination. After 6h, cultures were assessed for spa mRNA by RT-PCR, whilst SpA protein was measured by specific ELISA after 18h. When used alone, antibiotics (clindamycin, linezolid and tigecycline) or HNPs significantly reduced both SpA production and mRNA levels in ST30 and ST80 strains. When used in combination, HNPs and clindamycin, linezolid or tigecycline synergistically reduced SpA production (6-100-fold) and spa mRNA levels (4-20-fold) in ST80 and ST30 strains. In contrast, for USA300 strains, among all antibiotics, clindamycin alone reduced SpA production (3.5-fold), whereas with combined treatments including HNPs, only a slight reduction in SpA production (1.7-2.2-fold) was observed. In conclusion, antibiotics and AMPs do not modulate SpA expression in USA300, unlike in other CA-MRSA clones. This observation suggests that the virulence and successful spread of USA300 strains is associated with a specific regulatory network.

Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line.

High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM(+) cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM(-) cells). Both cell types were studied at genomic and functional levels. The DM(+) cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM(-) cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs.

Influence of CD34 cell selection on the incidence of mixed chimaerism and minimal residual disease after allogeneic unrelated donor transplantation.

Marrow transplantation from unrelated donors has been linked with an increased risk of graft-versus-host disease (GVHD). In an attempt to lower the risk of acute GVHD we used CD34 marrow cell selection for T cell depletion. Since T cell depletion has been linked to an increased risk of relapse and an increased risk of marrow failure, we used PCR amplification of minisatellite sequences to investigate donor cell engraftment and RT-PCR amplification of recurrent chromosomal translocations to investigate the residual disease post-transplant. Twenty-three patients who underwent BMT after positive selection of the CD34-positive cell population were studied. Results were then compared with those of 37 patients who underwent transplantation with unmanipulated marrow graft. Among the 23 patients who received CD34+ selected cell grafts, seven (30%) had evidence of full donor engraftment, 14 had evidence of residual recipient cells (61%), one had a non-take, and one autologous bone marrow recovery. Analysis of the chimaerism status post-transplant in 36 patients who received unmanipulated marrow grafts showed that 31 patients (86%) had evidence of full donor engraftment. The difference in the incidence of mixed chimaerism profile between patients who received unmanipulated marrow graft and those receiving CD34+ selected cell grafts was statistically significant (P< 0.01). Nine patients who received CD34+ selected cell grafts could be analysed for the presence of minimal residual disease post-transplant (one with t(9;22) acute lymphoblastic leukaemia and eight with CML). In the patient transplanted for a Ph-positive acute leukaemia, and in two out of the eight patients with CML, the search fora fusion transcript was consistently negative after transplantation. Among the six patients with evidence of residual disease, three patients also had a mixed chimaerism profile and were given donor lymphocyte infusions. Minimal residual disease study was performed post-transplant in 16 patients who received unmanipulated marrow grafts. In 10 of 14 patients with CML, and in two patients with acute leukaemia the search for a fusion transcript was consistently negative after transplantation. The difference in the incidence of minimal residual disease between patients who received an unmanipulated marrow graft and those receiving CD34+ selected cell grafts was not statistically significantly significant, but numbers of patients included in this analysis are still few. In conclusion, our study highlights the strong influence of graft manipulation on the incidence of mixed chimaerism after transplantation from an unrelated donor.

Detection of recipient cells after non T-cell depleted bone marrow transplantation for leukemia by PCR amplification of minisatellites or of a Y chromosome marker has a different prognostic value.

We used polymerase chain reaction amplification of minisatellite sequences (33.6.3, MS51, YNZ22) and of a Y chromosome-specific sequence (DYZ1) to document prospectively chimerism in 23 leukemia patients grafted with non T-cell depleted marrows from HLA-identical sibling donors. Twenty-two patients had a complete hematopoietic chimerism (within the sensitivity limit of the method used: 1%) early (about 1 month) after transplantation and one had detectable residual host cells (partial chimerism). These cells were still present after 8 months, and this patient relapsed 16 months after transplantation. Two patients with early complete chimerism relapsed 16 and 17 months after transplantation. Seven patients died from toxicity or infections and 13 are in clinical remission with a follow-up of 16 to 48 months. Nine male patients grafted with the marrow from a female donor were also studied by amplification of the DYZ1 marker (0.01% sensitivity). In all nine cases, residual male nucleated cells were detected early and up to 1 year after transplantation. These results suggest that the detection of persistent residual recipient cells above a 1% level might be predictive of relapse but that the detection of such cells in a 0.01-1% range is probably unrelated to relapse and does not seem to influence the outcome of the transplantation procedure.

Highly sensitive polymerase chain reaction methods show the frequent survival of residual recipient multipotent progenitors after non-T-cell-depleted bone marrow transplantation.

Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when analyzed by polymerase chain reaction (PCR) amplification of minisatellite sequences 33.6.3 and MS51 (0.1% to 1% sensitivity) were studied by the highly sensitive technique of PCR amplification of the Y-chromosome-specific DYZ1 sequence (0.01% sensitivity). Residual recipient male cells were detected in all peripheral blood samples collected within 1 year posttransplantation. These residual cells were present in both the lymphocyte and polymorphonuclear cell fractions when such a separation was performed by Ficoll gradient centrifugation and, for samples of 13 of 15 patients, at comparable levels in both fractions. In 3 samples collected from 3 patients 4 months or more posttransplantation, residual recipient cells were detected in the polymorphonuclear cell fraction but were present at a lower level or were undetectable in the lymphocyte fraction. These cells are of hematopoietic origin because they were detected at equivalent levels in whole blood and in B and T lymphocytes sorted with antibody-coated magnetic beads. They were not detected in samples collected more than 15 months posttransplantation for 6 of 7 patients. The persistence of residual recipient cells within 1 year posttransplantation is not restricted to male patients receiving a transplant from a female donor because they were also detected in 2 female patients using an allele-specific amplification method for the thyroid peroxydase gene that also has a high sensitivity (0.01%). Our results indicate that at least residual recipient myeloid progenitors and possibly totipotent hematopoietic stem cells may survive intensive pretransplant conditioning regimen and support a transient residual hematopoiesis of the host posttransplantation.

Donor B cell lymphoma of the brain after allogeneic bone marrow transplantation for acute myeloid leukemia.

We present a case of EBV-induced cerebral B cell lymphoproliferation of donor origin after HLA-matched allogeneic BMT for AML. The presentation was note-worthy as this 4-year-old girl was grafted after a conditioning regimen without irradiation, with an unmanipulated, HLA-matched graft. Furthermore, the cerebral mass developed long after reduction of immunosuppressive therapy following improvement of severe GVHD. To our knowledge, such cerebral lymphoproliferation, under these transplant conditions, has never been described.

Bone marrow transplantation for Fanconi anemia using low-dose cyclophosphamide/thoracoabdominal irradiation as conditioning regimen: chimerism study by the polymerase chain reaction.

Since 1976, patients grafted at the Hôpital Saint-Louis for Fanconi anemia (FA) without evidence of leukemic transformation have been given a uniform conditioning regimen that consisted of low-dose cyclophosphamide (Cy) and thoracoabdominal irradiation (TAI). The use of low-dose Cy raised the question of whether it is sufficient for the establishment of a complete hematopoietic chimerism in all patients. We therefore initiated a study of chimerism early during hematopoietic reconstitution after bone marrow transplantation (BMT) and thereafter in transplanted FA patients. Minisatellite probes were used after DNA amplification by the polymerase chain reaction (PCR). From July 1989 to October 1992, 24 consecutive patients underwent BMT for FA, 19 of whom were assessable for chimerism. Our results using this sensitive technique showed that, among these 19 patients, all but one successfully engrafted. Engraftment was complete early after BMT in 12. The persistence of a small proportion of recipient's cells was detected in six. This partial hematopoietic chimerism was demonstrated to be only transient in at least five of the six patients. The one patient who failed to engraft showed a recipient-type profile for circulating cells early posttransplantation, indicating autologous bone marrow recovery. A second graft in this patient was also rejected. For both transplantations, the patient was grafted from a matched, unrelated donor. Therefore, 17 of 17 patients successfully grafted and with complete follow up data presented complete hematopoietic chimerism, within the sensitivity limit of the method used. In conclusion, lowering the Cy dose in the conditioning regimen of patients with FA could still allow complete engraftment to occur, at least in patients with an identical sibling donor.

Short-term study of chimaerism after bone marrow transplantation for severe aplastic anaemia.

Chimaerism was studied early (2 weeks to 3 months) during haematopoietic reconstitution after bone marrow transplantation in 18 severe aplastic anaemia patients (acquired SAA: 14 patients; Fanconi anaemia: four patients). Fourteen patients received marrow from an identical sibling donor, one from the phenoidentical father and three from a matched unrelated donor. Peripheral blood cell DNA was first analysed by Southern blotting with a multilocus minisatellite probe (33.6.3) or a Y chromosome specific probe (pHY2.1). For all 14 patients grafted with a genotypically identical sibling donor, the post-graft DNA profile strictly matched the respective donor profile (minisatellite probe) or disclosed the Y chromosome specific band in the case of female patients grafted with a male donor. In contrast, for the one patient grafted in a mismatched situation and for two out of three patients grafted with a matched unrelated donor, the results indicated autologous bone marrow recovery. This difference between patients grafted with an identical sibling donor and those grafted in other situations is statistically significant (P less than 0.01). The 15 patients with circulating cells of donor origin were then studied by polymerase chain reaction amplification of the DNA samples. The three male patients with a female donor were studied by amplification of a Y chromosome specific sequence (DYZ1), allowing the detection of one male cell in 10(4) female cells. In all three cases, residual male nucleated cells were detected. The analysis was performed by amplification of the 33.6.3 minisatellite sequence for the 12 remaining patients. No residual recipient cells were detected within the sensitivity limit of the method which is 1% in that case. This suggests that detection of residual host cells depends on the sensitivity of the technique used.

Isoforms of the E2 molecule: D44 monoclonal antibody defines an epitope on E2 and reacts differentially with T cell subsets.

Human T cell rosetting with erythrocytes is clearly dependent on the CD2-CD58 interaction. We have previously demonstrated that other T cell molecules are involved in the rosette phenomenon, including the E2 molecule, a 32-kDa transmembrane glycoprotein. In this report we show that the D44 monoclonal antibody (mAb), previously shown to subdivide T cells into subpopulations with distinct functional repertoires and to identify 70% of lymphocytes from bronchoalveolar lavage from HIV+ patients, defined a new epitope on the E2 molecule. This was illustrated by the reactivity of the D44 mAb in Western blot experiments performed with the immunoaffinity purified E2 molecule. Moreover, double-labeling experiments showed that the E2 molecule exhibited varying epitopes when expressed in different cell types. The D44 and 12E7 epitopes were restricted to distinct subpopulations of T cells and, more importantly, the D44 expression was limited to the CD29+ population including the memory subset. The O662 and L129 epitopes are present on all T cells. Thus, the E2 molecule has both common and variable epitopes in its extracellular domain, and only the common epitopes seem to be involved in T cell adhesion processes.

The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product.

E2 is a 32 kd human T-cell surface glycoprotein involved in spontaneous rosette formation with erythrocytes. A 1.11 kb cDNA was isolated from a lambda gt11 expression library by screening with monoclonal antibodies directed against E2. The primary structure of E2, deduced from the nucleotide sequence of its gene, comprises 185 amino acids and is devoid of N-linked glycosylation sites. The E2 protein is rich in proline residues and displays an organization typical of an integral membrane protein. Northern blotting showed a good correlation between mRNA abundance, E2 surface density and the level of T cell differentiation. In fact, nucleotide sequencing revealed that E2 is the MIC2 gene product, previously identified with the 12E7 Mab. Xg(a-) female individuals have no E2 molecule on the surface of their red cells, in contrast with Xg(a+) individuals, but have the molecule in their cytoplasm, in the form of the 28 kd precursor. These findings show that the E2 antigen, a cell surface molecule involved in T cell adhesion processes, is the product of the MIC2 gene, the only pseudoautosomal gene to be described in man.

The biochemical characterization of E2, a T cell surface molecule involved in rosettes.

We have previously identified a molecule on the T cell surface, which, in addition to CD2 is involved in the rosette phenomenon. This is a 32-kDa single polypeptide chain which we have termed E2. The studies reported here show striking patterns on the glycosylation status of E2. It is a heavily sialylated and glycosylated molecule, the sugar moieties accounting for almost half of its relative molecular mass (Mr). It carries no N-linked sugar residues, only O-linked oligosaccharides. Despite heavy glycosylation, the molecule appears to behave homogeneously on gel electrophoresis, both in terms of Mr and pI. Neuraminidase treatment of E2 lowered its Mr to 28 kDa; this was further decreased to 18 kDa after removal of O-linked sugar residues by treatment with O-glycanase. An identical reduction in size was observed after treatment with trifluoromethane sulfonic acid, showing that the molecule carries no detectable N-linked sugar residues. Moreover, endoglycosidase F and endoglycosidase H treatment of either the immunoprecipitates from 125I surface-labeled thymocytes, or of a purified preparation of E2, did not reduce the Mr of E2, nor did tunicamycin treatment of T cells. Two-dimensional gel electrophoresis revealed two discrete spots of acidic pI (4.4 and 4.6) that were still seen after neuraminidase treatment, though they had moderately shifted. Pulse-chase experiments revealed a single 28-kDa precursor form that could have been the unsialylated molecule. Finally, sequencing 14 amino acid residues of the N-terminal side revealed no homology with known proteins. Since the sugar moieties of adhesion protein could play an important role, the results obtained in this study will prove valuable to our understanding of the role exerted by the E2 molecule.

A T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes.

Increasing evidence indicates that rosettes which spontaneously from between human T cells and E might be of physiologic relevance. We show here that another T cell-surface molecule than CD2 is involved in rosette formation. Four mAb have been obtained reacting with human T cells that block rosettes with E from many species, including autologous cells. They react with a molecule, we termed E2, which is actively synthetized by T and monocytic cells. Immunoprecipitation revealed a major 32-kDa band. Immunoblots revealed a major 32-kDa band and a minor 20-kDa band. This molecule was detected on all T cells tested--and present at high densities on corticothymocytes, but at low densities on medullary thymocytes. It was also found on monocytes but not on B cells. However B-CLL cells did carry this molecule. E2 molecules were also detected on nonhematologic cells. Together with the recent evidence that 3 molecules from the erythrocyte surface are also involved for rosettes, intricate molecular interactions would account for adhesion of T cells to autologous E and possibly autologous nucleated cells.

Role in T-cell activation for HLA class I molecules from accessory cells: further distinction between activation signals delivered to T cells via CD2 and CD3 molecules.

The immunological function of major histocompatibility complex molecules, including HLA class I molecules, is to present antigens and/or their processed peptides to various lymphocyte subpopulations. Thus, they play a pivotal role in regulatory interactions between cells of the immune system, which can result in the activation and function of T cells. We looked for a role of major histocompatibility complex molecules during T-cell activation induced by monoclonal antibody (mAb) or combinations of mAb recognizing the two well-characterized T-cell surface molecules CD3 and CD2. To activate T-cell peripheral blood lymphocytes, we used a CD3 mAb or two different pairs of CD2 mAb, CD2 "GT2 + T11(1)" and CD2 "D66 + T11(1)," which, as we have previously shown, deliver different signals of activation to T cells. Anti-HLA class I mAb blocked the activation induced by CD3 mAb or by CD2 GT2 + T11(1), but it did not block activation induced by CD2 D66 + T11(1). We observed this pattern of inhibition according to the stimulus used to activate T cells both when the anti-HLA class I mAbs were added to cultures of whole peripheral blood mononuclear cells and when they were fixed to monocytes only. In the latter case, purified monocytes were first incubated with the anti-HLA mAb (whether whole immunoglobulin or Fab fragment) and then fixed with paraformaldehyde before culture with autologous purified T cells. Anti-HLA class I fixed on monocytes prevented both interleukin 2 (IL-2) receptor expression and IL-2 synthesis on T cells. The inhibitory effects of anti-class I mAb bound to monocytes were not reversed by adding large amounts of recombinant IL-2 or recombinant IL-1, a finding consistent with the observations that accessory cells surface components can fully complement the signals directly delivered to T cells by CD2 or CD3 mAb. We conclude that HLA class I from accessory cells plays an important role in the early phase of T-cell activation when direct contacts between accessory cells and T cells are required.

[Implication of HLA class I molecule from monocyte in T-cell activation by CD2 or CD3 monoclonal antibodies]. / Implication des molécules HLA de classe I du monocyte lors de l'activation des cellules T par des anticorps monoclonaux CD2 ou CD3.

The role of monocytes in human T-cell activation by monoclonal antibodies (mAb) recognizing CD3 molecule or by 2 mAb pairs directed against different epitopes of CD2 "GT2+T11(1)" or "D66+T11(1)" has been studied. It appears that HLA-cl I molecules from monocytes are involved in the early activation phase of T-cells stimulated by CD3 or CD2 when direct contacts between T-cells and monocytes are required. Thus, pretreatment of monocytes with HLA-cl I mAb inhibited IL 2 receptor appearance and IL 2 synthesis on T-cells stimulated by CD3 mAb or CD2 "GT2+T11(1)" mAb pair.

A unique epitope on the CD2 molecule defined by the monoclonal antibody 9-1: epitope-specific modulation of the E-rosette receptor and effects on T-cell functions.

Monoclonal antibody (MoAb) 9-1 shows unique binding properties to CD2 resulting in peculiar epitope specific changes. 9-1 shows competitive binding with MoAb to D66 epitope, and gives a similar staining pattern with T-cell populations, at a low density on resting T cells, and high density on thymocytes and activated T cells. However, 9-1 has the opposite effect on anti-D66 MoAbs on rosette formation, namely, 9-1 increases the stability of rosettes, but 9-1 plus anti-mouse Ig bound to T-cell surface blocks rosettes. 9-1 plus anti-mouse Ig, like anti-D66 MoAbs, induces further appearance of D66 and 9-1 epitopes but, contrary to anti-D66, induces appearance of T11(3) epitopes. Thus, binding 9-1 results in unique "epitope-specific modulation" events that are not solely artificial, but appear to mimic events naturally occurring during T-cell differentiation/activation. The effects of binding 9-1 on T-cell functions also display peculiarities. 9-1, like anti-D66 MoAbs, activates T cells when added in combination with anti-9.6/T11(1) MoAbs but not with anti-T11(3). To obtain full activation, monocytes are required; however, adding 9-1 alone do not inhibit specific T-cell cytotoxicity contrary to anti-D66 or anti-9.6/T11(1), although 9-1 inhibits NK activity of peripheral cells. Given the apparent complexities of the functions exerted by CD2, these data show that definite conformational changes or reorientation, which would be naturally produced by soluble and/or cell surface ligand(s), would be key events in determining how CD2 will influence T-cell functions.

Heterogeneity of the first cluster of differentiation: characterization and epitopic mapping of three CD1 molecules on normal human thymus cells.

In humans, the presence of two non-HLA class 1-like molecules, whose expression, similar to murine Tla, is restricted to cortical thymocytes, has been shown with monoclonal antibodies defining the first cluster of differentiation (CD1). We report here with the use of 12 anti-CD1 antibodies and a combination of technical approaches, the characterization of a third CD1 molecule. We show that we can presently define seven different epitopes on the three CD1 molecules: four epitopes are restricted to the 49,000 dalton molecule, two epitopes to the 45,000 dalton molecule, and one epitope to the 43,000 dalton molecule. We show that the association of the newly identified 45,000 dalton heavy chain with human beta2-microglobulin is weak. In addition we show the presence of a fourth non-HLA class I molecular species on the surface of normal human thymus cells.

[Fall in the levels of circulating lymphoblasts caused by intravenous administration of the A50 monoclonal antibody in a patient with acute T-cell lymphoblastic leukemia. A step in the demonstration of serotherapy with monoclonal antibodies]. / Chute du taux des lymphoblastes circulants par administration intra-veineuse de l'anticorps monoclonal A50 chez un malade ayant une leucémie aiguë lymphoblastique à cellules T. Une étape dans la mise au point de la sérothérapie par les anticorps monoclonaux.

In an 8-year-old boy with acute lymphoblastic leukaemia not previously treated intravenous administration of a single 8 mg dose of a monoclonal antibody that recognizes an epitope restricted to the surface of mature T-cells resulted, within 20 hours, in a fall in circulating lymphoblasts from 200 000 to 70 000. No adverse clinical or biological reaction was detected and no antigenic modulation occurred at lymphoblast surface. This observation constitutes a first step in the complex development of effective serotherapy for malignant diseases, using monoclonal antibodies.

Phenomenon of human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies. "Modulation" of a partially hidden epitope determining the conditions of interaction between T cells and erythrocytes.

Anti-D66 is a monoclonal antibody able to inhibit E-rosette formation of T cells both at 4 degrees C and at 37 degree C but that does not inhibit T cell rosette formation with neuraminidase or 2-amino-ethylisothiouronium bromide (AET)-pretreated E. As demonstrated by capping experiments, it defines an epitope, D66, that is directly involved in E-rosette formation. D66 is distinct from the epitope defined by 9.6 because 9.6, a previously defined "pan-T" monoclonal antibody, inhibits E(AET) rosette formation and because no cross-blocking occurred between both antibodies fixation. However, 9.6 and D66 are carried by the same molecule, as demonstrated by sequential immunoprecipitation assays performed on two different T cell lines. On the thymocyte surface, also, 9.6 and D66 are most probably carried by the same molecule, as indicated by cocapping and colysostripping experiments. D66 is present at higher densities on thymocytes and activated T cells than on peripheral blood T cells. Investigation of numerous T cell populations, both normal and malignant, showed a straightforward correlation between elevated D66 density and ability to form 37 degrees C stable E-rosettes. Neuraminidase treatment of thymocytes and peripheral blood lymphocytes forming E-rosettes unmasked a large fraction of D66 not readily accessible on their surface. These hidden D66 epitopes appear to be responsible for a surprising observation: the ability of anti-D66 to inhibit E-rosette formation could be totally reversed by fixation on anti-D66 of an antibody to mouse immunoglobulin or an Fab fragment anti-mouse immunoglobulin. This would induce microdisplacement with emergence of hidden D66, as documented by fluorometric studies. Finally, malignant T cells with a differentiative status of mature T cells, but forming no (or low numbers of) E-rosettes, could be induced both to display D66 and to form E-rosettes by neuraminidase treatment.