hMENA(11a) contributes to HER3-mediated resistance to PI3K inhibitors in HER2-overexpressing breast cancer cells.

Human Mena (hMENA), an actin regulatory protein of the ENA/VASP family, cooperates with ErbB receptor family signaling in breast cancer. It is overexpressed in high-risk preneoplastic lesions and in primary breast tumors where it correlates with HER2 overexpression and an activated status of AKT and MAPK. The concomitant overexpression of hMENA and HER2 in breast cancer patients is indicative of a worse prognosis. hMENA is expressed along with alternatively expressed isoforms, hMENA(11a) and hMENAΔv6 with opposite functions. A novel role for the epithelial-associated hMENA(11a) isoform in sustaining HER3 activation and pro-survival pathways in HER2-overexpressing breast cancer cells has been identified by reverse phase protein array and validated in vivo in a series of breast cancer tissues. As HER3 activation is crucial in mechanisms of cell resistance to PI3K inhibitors, we explored whether hMENA(11a) is involved in these resistance mechanisms. The specific hMENA(11a) depletion switched off the HER3-related pathway activated by PI3K inhibitors and impaired the nuclear accumulation of HER3 transcription factor FOXO3a induced by PI3K inhibitors, whereas PI3K inhibitors activated hMENA(11a) phosphorylation and affected its localization. At the functional level, we found that hMENA(11a) sustains cell proliferation and survival in response to PI3K inhibitor treatment, whereas hMENA(11a) silencing increases molecules involved in cancer cell apoptosis. As shown in three-dimensional cultures, hMENA(11a) contributes to resistance to PI3K inhibition because its depletion drastically reduced cell viability upon treatment with PI3K inhibitor BEZ235. Altogether, these results indicate that hMENA(11a) in HER2-overexpressing breast cancer cells sustains HER3/AKT axis activation and contributes to HER3-mediated resistance mechanisms to PI3K inhibitors. Thus, hMENA(11a) expression can be proposed as a marker of HER3 activation and resistance to PI3K inhibition therapies, to select patients who may benefit from these combined targeted treatments. hMENA(11a) activity could represent a new target for antiproliferative therapies in breast cancer.

Neonatal-mouse infectivity of intact Cryptosporidium parvum oocysts isolated after optimized in vitro excystation.

We reexamined the finding of Neumann et al. that intact Cryptosporidium parvum oocysts obtained after in vitro excystation were infectious for neonatal CD-1 mice. We used both established excystation protocols and our own protocol that maximized excystation. Although intact oocysts isolated after any of three protocols were infectious for neonatal CD-1 mice, the infectivity of intact oocysts isolated with our optimized excystation protocol was significantly lower than the infectivity of intact oocysts isolated after established protocols or from fresh oocysts. Excystation should not be considered a valid measure of C. parvum viability, given that it is biologically implausible for oocysts to be nonviable and yet infectious.

Cytotoxic T-lymphocyte responses in melanoma through in vitro stimulation with the Melan-A peptide analogue A27L: a qualitative analysis.

Modifications in tumour antigen-derived epitopes that stabilize the major histocompatibility complex (MHC)-peptide complex result in enhanced stimulatory capacity and improved immunogenicity of the altered peptide. These epitope analogues are attractive candidates for the development of peptide-based vaccine trials. Any modification, however, in tumour antigens may induce T-cell responses that could either fail to react against the naturally occurring peptides or represent only a subset of the total antigen-specific repertoire. In the present study, we performed a critical analysis of the ability of cytotoxic T-lymphocyte (CTL) clones, derived from two melanoma patients through stimulation with the A27L peptide analogue, to cross-react with the naturally processed Melan-A/MART-1 (Melan-A) peptides in terms of T-cell receptor (TCR) affinity, functional avidity and fine antigen specificity. We found that all the A27L-specific clones analysed possessed a very low avidity for the natural Melan-A peptides, and that their binding affinity for human leukocyte antigen (HLA) tetramers complexed with both the modified and the natural Melan-A peptides did not strictly correlate with their functional avidity. We also observed that these clones were able to cross-recognize both natural Melan-A peptides in one patient, but only one peptide in the second patient. We discuss the capability of the A27L peptide analogue to stimulate all the available Melan-A-specific repertoire.

Specific cytotoxic T lymphocyte responses against Melan-A/MART1, tyrosinase and gp100 in vitiligo by the use of major histocompatibility complex/peptide tetramers: the role of cellular immunity in the etiopathogenesis of vitiligo.

Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

Kinetics of GATA-3 gene expression in early polarizing and committed human T cells.

Different transcription factors have been shown to control the transition of naive T cells into T helper 1 (Th1)/Th2 subsets. The T-cell-specific transcription factor GATA-3 is known to be selectively expressed in murine developing Th2 cells and to exert a positive action on Th2-specific cytokine production. Investigating GATA-3 gene regulation in human T cells we have found that naive T cells highly express GATA-3, and during early T2 or T1 polarization, respectively, they either maintain or quickly down-regulate expression. In developing T2 cells, as well as in committed Th2 cell lines and clones, we found a positive correlation among GATA-3, interleukin (IL)-5 and IL-4 gene expression kinetics, supporting the positive action of GATA-3 on Th2-specific cytokine production. A possible relationship between GATA-3 gene expression and the down-regulation of the IL-12 receptor (beta2-chain; IL-12Rbeta2) gene was evident only in the early phases of T2 polarization (within 24 hr), and not demonstrated at later times. During T-cell commitment the presence of IL-4 in the culture was essential to maintain or enhance GATA-3 transcription, while IL-12 was not necessary for full repression of GATA-3. Finally, we showed selective GATA-3 up-regulation in human Th2 cell lines and clones and the maintainance of a low basal level of GATA-3 expression in Th1 cells upon activation.

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients.

While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.

Increased frequency of RAG-expressing, CD4(+)CD3(low) peripheral T lymphocytes in patients with defective responses to DNA damage.

Accumulating evidence indicates that peripheral lymphocyte variants with altered antigen receptor expression may be capable of expressing recombination-activating genes (RAG). We and others recently observed functional RAG gene products in mature T cells with defective TCR expression (MacMahan and Fink, Immunity 1998. 9: 637 - 647; Lantelme et al., J. Immunol., 2000. 164: 3455 - 3459). Here, the association between TCR expression and RAG activity was assessed further in lymphocytes from patients with defective responses to DNA damage. We show that T cells with altered TCR surface expression are present in increased numbers in these patients and that they express RAG genes. The finding of RAG gene expression by TCR variants suggests the possibility that secondary V(D)J rearrangements could be induced in these cells to rescue their defective phenotype and cellular function. Moreover, as V(D)J recombination has been implicated in chromosome translocations involving antigen receptor genes, we discuss a possible relationship between altered TCR expression, RAG activity and the frequent lymphoma-specific translocations observed in these patients.

Cutting edge: recombinase-activating gene expression and V(D)J recombination in CD4+CD3low mature T lymphocytes.

The recombinase-activating genes, RAG-1 and RAG-2, can be expressed by a subset of B cells within germinal centers, where they mediate secondary V(D)J rearrangements. This receptor revision mechanism could serve either receptor diversification or tolerance-induced functions. Alternatively, it might rescue those cells the receptors of which have been damaged by somatic mutation. Less is known about the occurrence of similar mechanisms in T cells. Here we show that mature T cells with defective TCR surface expression can express RAG genes and are capable of initiating secondary V(D)J rearrangements. The possibility that a cell rescue mechanism based on the generation of a novel Ag receptor might be active in peripheral T cells is envisaged.

Selection and mapping of replication origins from a 500-kb region of the human X chromosome and their relationship to gene expression.

In higher eukaryotes the mechanism controlling initiation of DNA replication remains largely unknown. New technologies are needed to shed light on how DNA replication initiates along the genome in specific regions. To identify the human DNA sequence requirements for initiation of replication, we developed a new method that allows selection of replication origins starting from large genomic regions of human DNA. We repeatedly isolated 15 new putative replication origins (PROs) from a human DNA region of 500 kb in which 17 genes have previously been characterized. Fine-mapping of these PROs showed that DNA replication can initiate at many specific points along actively transcribed DNA in the cell lines used for our selection. In conclusion, in this paper we describe a new method to identify PROs that suggests that the availability of initiation sites is dependent on the transcriptional state of the DNA.

Depression and anxiety in Parkinson's disease: possible effect of genetic variation in the serotonin transporter.

Recently, a functional polymorphism in the promoter region of the serotonin transporter gene has been linked to anxiety. In cell culture, the short allele of this polymorphism synthesizes less serotonin transporter, resulting in a reduction of the removal of serotonin from the synaptic cleft. This pilot study examines depression and anxiety in Parkinson's disease patients as a function of the variation in this polymorphism. Thirty-two patients were genotyped and then blindly administered the Hamilton Depression and Anxiety Scales. Clinical data on the neurologic features of the disease were also gathered. Patients with the short allele of the serotonin transporter promotor scored significantly higher on both the depression and anxiety measures. There were no differences between groups for any neurologic variable. Patients with the short allele were more likely to have scores for anxiety and depression that indicated "caseness." This study suggests that the short allele of the serotonin transporter gene may represent a significant risk factor for the development of anxiety and depression in Parkinson's disease patients.

Quetiapine as an alternative to clozapine in the treatment of dopamimetic psychosis in patients with Parkinson's disease.

There are many difficulties associated with the late stages of Parkinson's disease (PD), but psychosis and agitation may be the most disturbing for both patients and care givers, and often precipitate the pivotal decision for long-term nursing home placement. While the addition of antipsychotic drugs or the withdrawal of antiparkinsonian drugs may improve the behavioral problem, these strategies usually worsen the motor difficulties. Clozapine has been studied in PD for over a decade, and while it appears to be effective, there are safety and tolerability concerns associated with it. In addition, in New Jersey, Medicaid no longer pays for the home blood draws that are required for home-bound patients. This led to a situation in which we had patients who needed to stop clozapine and begin an alternative therapy. Because quetiapine seems particularly well suited to patients with PD based on in vitro and in vivo studies we have begun to try this medication in PD patients who need to stop clozapine. This article reports three case histories of patients with PD, confusion and dopamimetic psychosis who had been previously managed with clozapine and who were successfully switched to quetiapine. At doses from 12.5 to 150 mg/day quetiapine was well tolerated, resulting in behavioral improvement and no real increase in parkinsonism. These case histories raise the possibility that quetiapine may represent a viable alternative to clozapine in PD patients with dopamimetic psychosis and behavioral disturbances.

Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells.

When naive T lymphocytes are activated and differentiate into memory/effector cells, they down-regulate receptors for constitutive chemokines such as CXCR4 and CCR7 and acquire receptors for inflammatory chemokines such as CCR3, CCR5 and CXCR3, depending on the Th1/Th2 polarization. This switch in chemokine receptor usage leads to the acquisition of the capacity to migrate into inflamed tissues. Using RNase protection assays, staining with specific antibodies, and response to recombinant chemokines, we now show that following TCR stimulation, memory/effector T cells undergo a further and transient switch in receptor expression. CCR1, CCR2, CCR3, CCR5, CCR6 and CXCR3 are down-regulated within 6 h, while CCR7, CCR4, CCR8 and CXCR5 are up-regulated for 2 to 3 days. Up-regulation of CCR7 following TCR stimulation was observed also among resting peripheral blood T cells and required neither co-stimulation nor exogenous IL-2. On the other hand IL-2 down-regulated CXCR5, up-regulated CCR8 and facilitated the recovery of CCR3 and CCR5. Upon TCR stimulation, Th1 and Th2 cells produced comparable sets of chemokines, including RANTES, macrophage inflammatory protein-1beta, I-309, IL-8 and macrophage-derived chemokine, which may modulate surface chemokine receptors and contribute to cell recruitment at sites of antigenic recognition. Altogether these results show that following TCR stimulation effector/memory T cells transiently acquire responsiveness to constitutive chemokines. As a result, T cells that are activated in tissues may either recirculate to draining lymph nodes or migrate to nearby sites of organized ectopic lymphoid tissues.

Distinct patterns and kinetics of chemokine production regulate dendritic cell function.

Dendritic cells (DC) have been showed to both produce and respond to chemokines. To understand how this may impact on DC function, we analyzed the kinetics of chemokine production and responsiveness during DC maturation. After stimulation with LPS, TNF-alpha or CD40 ligand, the inflammatory chemokines MIP-1alpha, MIP-1beta and IL-8 were produced rapidly and at high levels, but only for a few hours, while RANTES and MCP-1 were produced in a sustained fashion. The constitutive chemokines TARC, MDC and PARC were expressed in immature DC and were up-regulated following maturation, while ELC was produced only at late time points. Activated macrophages produced a similar spectrum of chemokines, but did not produce TARC and ELC. In maturing DC chemokine production had different impact on chemokine receptor function. While CCR1 and CCR5 were down-regulated by endogenous or exogenous chemokines, CCR7 levels gradually increased in maturing DC and showed a striking resistance to ligand-induced down-regulation, explaining how DC can sustain the response to SLC and ELC throughout the maturation process. The time-ordered production of inflammatory and constitutive chemokines provides DC with the capacity to self-regulate their migratory behavior as well as to recruit other cells for the afferent and efferent limb of the immune response.

Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results.

Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.

Stem cell enumeration in cord blood vs bone marrow and peripheral blood.

Recent reports have suggested that the total number of autologous or allogeneic hematopoietic stem cell (HSC) infused after high-dose chemotherapy might predict survival, post-transplant morbidity and rate of hematopoietic engraftment. However, HSC capable of long-term multilineage potential are still poorly defined, and tools for accurate and reproducible HSC enumeration are highly warranted.

Control of stem cell proliferation and differentiation to achieve stable gene transfer and expression.

Although the hematopoietic stem cell (HSC) seems to be a convenient target for gene therapy, data from human clinical trials have so far shown low levels of gene transduction. The new model of human stem cells, immunodeficient mice repopulating cells (SRC), has similarly demonstrated that SRC were rarely transduced by protocols used in past studies. Cytokines such as stem cell factor and interleukin-3, used so far to obtain cell proliferation in transduction protocols, might induce HSC to differentiate and impair their repopulating potential. In this scenario, there is a need for new gene transfer protocols associated with minimal cell differentiation and stable expression of the transduced gene in the majority of mature cells generated from transduced HSC.

Hematopoietic stem cells from different sources: biological and technical aspects.

Hematopoietic stem cell (HSC) enumeration is crucial to predict the engraftment potential of a given HSC collection, and currently involves the surrogate count of nucleated cells, CFU or CD34+ cells. However, there is raising evidence that CFU are HSC involved in short-term but not in long-term reconstitution, and that only a small fraction of all CD34+ cells have long term multilineage engraftment potential. In this regard, there is evidence that cord blood (CB), bone marrow (BM) and peripheral blood (PB) derived HSC are highly heterogeneous for a number of antigens useful for HSC enumeration by flow cytometry. Moreover, there is a raising evidence that a CD34 human HSC might exist. The CD34 HSC has been already described in animals and in human Hoechst 33342 negative HSC. This notwithstanding, clinical data have clearly demonstrated that purified allogeneic CD34+ cells can reconstitute the myeloid and the lymphoid lineages in myeloablated recipients. In the lack of a suitable marker for CD34 HSC enumeration, it is hard to predict the role of CD34 HSC in hematopoietic reconstitution after transplantation. On the other hand, these cells might be a better target for HSC expansion and gene transfer.

Multilineage long-term engraftment potential of drug-resistant hematopoietic progenitors.

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38-, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.

Effect of topical application with capsaicin on skin responses to bradykinin and histamine in man.

Pre-treatment with topical capsaicin is known to induce neuropeptide depletion from sensory nerve endings and it is a useful pharmacological tool to evaluate the contribution of these nerves to skin injury and inflammation. To investigate the relative contribution of sensory neural stimulation to the action of bradykinin and histamine, a randomized, double-blind study has been undertaken evaluating the effect of topical capsaicin pre-treatment on the responses to intradermal injections of both agonists in 12 healthy volunteers. Capsaicin pre-treatment caused significant inhibition of the immediate mean flare responses (95% CI) to both bradykinin (from 51.5 [39.7-63.3] mm2 to 16.2 [8.0-24.5] mm2) (P < 0.01) and histamine (from 108.4 [80.4-136.4] mm2 to 52.3 [37.1-67.1] mm2) (P < 0.01). Topical capsaicin elicited a significant inhibition of the weal response induced by histamine, the mean weal area being reduced from 14.8 (12.6-17.0) mm2 to 12.1 (10.1-14.1) mm2 (P < 0.05). In addition, the effect of topical capsaicin was to completely inhibit the bradykinin induced weal response compared to control, the mean weal area (95% CI) being reduced from 13.4 (11.4-15.4) mm2 to 8.2 (5.3-11.0) mm2 (P < 0.01). Our findings show that repeated topical application with capsaicin led to a significant reduction of the skin responses to intradermal injections with both agonists, and particularly with bradykinin. The weal responsiveness to bradykinin may entirely follow neuropeptide release from sensory nerves within the skin and the same applies to the flare response, although this is not completely inhibited by topical application with capsaicin.