In vitro effect of amifostine on haematopoietic progenitors exposed to carboplatin and non-alkylating antineoplastic drugs: haematoprotection acts as a drug-specific progenitor rescue.

We evaluated the protective ability of amifostine on peripheral blood mononuclear cell (PBMC)-derived colony-forming unit (CFU) and PB CD34+ cells which were previously exposed in vitro to etoposide, carboplatin, doxorubicin and taxotere. Amifostine pretreatment protected PBMC-derived CFU from the toxic effect of etoposide, carboplatin and taxotere. A significant detrimental effect was exerted by amifostine on the growth of doxorubicin-treated PBMC-derived CFU. Liquid cultures of PB CD34+ cells reproduced faithfully the effects observed on growth of PBMC-derived CFU and confirmed amifostine chemoprotection against etoposide and carboplatin with its detrimental effect on doxorubicin-treated progenitors. Combining the data of viable cell count, cytometric estimation of apoptosis, cell cycle and viable cell replication rate, we found that amifostine protects from etoposide and carboplatin toxicity mainly through a mechanism of cell rescue. Conversely, the detrimental effect of amifostine on the growth of doxorubicin-treated PB CD34+ cells is apparently due to an increased G2/M arrest. In conclusion, amifostine protects haematopoietic progenitors from etoposide, carboplatin and taxotere. Progenitor rescue is the mechanism through which amifostine reduced etoposide and carboplatin toxicity

Collagen-specific T-cell repertoire in blood and synovial fluid varies with disease activity in early rheumatoid arthritis

Type II collagen is a DR4/DR1 restricted target of self-reactive T cells that sustain rheumatoid arthritis. The aim of the present study was to analyze the T-cell receptor repertoire at the onset of and at different phases in rheumatoid arthritis. We used the CDR3 BV-BJ spectratyping to study the response to human collagen peptide 261-273 in 12 patients with DR4+ rheumatoid arthritis (six at the onset of disease and six during the course of disease) and in five healthy DR4+ relatives. The collagen-specific T-cell repertoire is quite restricted at the onset of disease, involving approximately 10 rearrangements. Within the studied collagen-specific rearrangements, nearly 75% is shared among patients. Although the size of the repertoire used by control individuals is comparable to that of patients, it is characterized by different T-cell receptors. Part of the antigen-specific T-cell repertoire is spontaneously enriched in synovial fluid. The specific T-cell repertoire in the periphery was modulated by therapy and decreased with the remission of the disease. Failure of immunoscopy to detect this repertoire was not due to suppression of collagen-driven proliferation in vitro by CD4+ CD25+ T cells. Clinical relapse of the disease was associated with the appearance of the original collagen-specific T cells. The collagen-specific T-cell receptor repertoire in peripheral blood and synovial fluid is restricted to a limited number of rearrangements in rheumatoid arthritis. The majority of the repertoire is shared between patients with early rheumatoid arthritis and it is modulated by therapy

Androgen receptors and hormone sensitivity of a human prostatic cancer cell line (PC-3) are modulated by natural beta-interferon

Androgen recptors are expressed at a low level in the cell line PC-3, which does not respond to either androgens or antiandrogens. If these cells are exposed to natural beta-interferon (β-IFN) a reduction in cell growth and an increase in androgen receptors, evaluated by both biochemical and immunocytochemical techniques, occur. This increase seems not to be related to a selective block of PC-3 in any phase of the cell cycle. Pretreatment with β-IFN determines in PC-3 cells a partial responsiveness to the androgen dihydrotestosterone as reflected by the increase in cell number. Moreover, the antiandrogen hydroxyflutamide shows agonistic properties by increasing the cell number of PC-3 cells pre-exposed to β-IFN. When the antiandrogen is tested in combination with interferon, it produces a reduction in the β-IFN-induced inhibition of cell growth. It is not known whether these unexpected effects are due to the increase in androgen receptors or to other mechanisms

Blood serum amyloid A as potential biomarker of pembrolizumab efficacy for patients affected by advanced non-small cell lung cancer overexpressing PD-L1: results of the exploratory "FoRECATT" study

Background: Identifying the patients who may benefit the most from immune checkpoints inhibitors remains a great challenge for clinicians. Here we investigate on blood serum amyloid A (SAA) as biomarker of response to upfront pembrolizumab in patients with advanced non-small-cell lung cancer (NSCLC). Methods: Patients with PD-L1 ≥ 50% receiving upfront pembrolizumab (P cohort) and with PD-L1 0-49% treated with chemotherapy (CT cohort) were evaluated for blood SAA and radiological response at baseline and every 9 weeks. Endpoints were response rate (RR) according to RECIST1.1, progression-free (PFS) and overall survival (OS). The most accurate SAA cut-off to predict response was established with ROC analysis in the P cohort. Results: In the P Cohort (n = 42), the overall RR was 38%. After a median follow-up of 18.5 months (mo), baseline SAA ≤ the ROC-derived cut-off (29.9 mg/L; n = 28/42.67%) was significantly associated with higher RR (53.6 versus 7.1%; OR15, 95% CI 1.72-130.7, p = 0.009), longer PFS (17.4 versus 2.1 mo; p < 0.0001) and OS (not reached versus 7.2mo; p < 0.0001) compared with SAA > 29.9 mg/L. In multivariate analysis, low SAA positively affects PFS (p = 0.001) and OS (p = 0.048) irrespective of ECOG PS, number of metastatic sites and pleural effusion. SAA monitoring (n = 40) was also significantly associated with survival endpoints: median PFS 17.4 versus 2.1 mo and median OS not reached versus 7.2 mo when SAA remained low (n = 14) and high (n = 12), respectively. In the CT Cohort (n = 30), RR was not affected by SAA level (p > 0.05) while low SAA at baseline (n = 17) was associated with better PFS (HR 0.38, 95% CI 0.16-0.90, p = 0.006) and OS (HR 0.25, 95% CI 0.09-0.67, p < 0.001). Conclusion: Low SAA predicts good survival outcomes irrespective of treatment for advanced NSCLC patients and higher likelihood of response to upfront pembrolizumab only. The strong prognostic value might be exploited to easily identify patients most likely to benefit from immunotherapy. A further study (FoRECATT-2) is ongoing to confirm results in a larger sample size and to investigate the effect of SAA on immune response in vitro assays

A novel assay of antimycobacterial activity and phagocytosis by human neutrophils

SummaryDespite abundant evidence that neutrophils arrive early at sites of mycobacterial disease and phagocytose organisms, techniques to assay phagocytosis or killing of mycobacteria by these cells are lacking. Existing assays for measuring the antimycobacterial activity of human leukocytes require cell lysis which introduces new bioactive substances and may be incomplete. They are also time-consuming and carry multiple risks of inaccuracy due to serial dilution and organism clumping. Flow cytometric techniques for measuring phagocytosis of mycobacteria by human cells have failed to adequately address the effects of organism clumping, quenching agents and culture conditions on readouts.Here we present a novel in-tube bioluminescence-based assay of antimycobacterial activity by human neutrophils. The assay yields intuitive results, with improving restriction of mycobacterial bioluminescence as the ratio of cells to organisms increases. We show that lysis of human cells is not required to measure luminescence accurately.We also present a phagocytosis assay in which we have minimised the impact of mycobacterial clumping, investigated the effect of various opsonisation techniques and established the correct usage of trypan blue to identify surface-bound organisms without counting dead cells. The same multiplicity of infection and serum conditions are optimal to demonstrate both internalisation and restriction of mycobacterial growth

Tamoxifen induces oxidative stress and apoptosis in oestrogen receptor-negative human cancer cell lines

Recent data have demonstrated that the anti-oestrogen tamoxifen (TAM) is able to facilitate apoptosis in cancer cells not expressing oestrogen receptor (ER). In an attempt to identify the biochemical pathway for this phenomenon, we investigated the role of TAM as an oxidative stress agent. In two ER-negative human cancer cell lines, namely T-leukaemic Jurkat and ovarian A2780 cancer cells, we have demonstrated that TAM is able to generate oxidative stress, thereby causing thiol depletion and activation of the transcriptional factor NF-κB. As described for other oxidative agents, TAM was able to induce either cell proliferation or apoptosis depending on the dose. When used at the lowest dose tested (0.1 μM), a slight proliferative effect of TAM was noticed in terms of cell counts and DNA synthesis rate, whereas at higher doses (10 μM) a consistent occurrence of apoptosis was detected. Importantly, the induction of apoptosis by TAM is not linked to down-regulation or functional inactivation by phosphorylation of the antiapoptotic bcl-2 protein. © 1999 Cancer Research Campaig

The Role of Regulatory T Cells in Cancer

There has been an explosion of literature focusing on the role of regulatory T (Treg) cells in cancer immunity. It is becoming increasingly clear that Treg cells play an active and significant role in the progression of cancer, and have an important role in suppressing tumor-specific immunity. Thus, there is a clear rationale for developing clinical strategies to diminish their regulatory influences, with the ultimate goal of augmenting antitimor immunity. Therefore, manipulation of Treg cells represent new strategies for cancer treatment. In this Review, I will summarize and review the explosive recent studies demonstrating that Treg cells are increased in patients with malignancies and restoration of antitumor immunity in mice and humans by depletion or reduction of Treg cells. In addition, I will discuss both the prognostic value of Treg cells in tumor progression in tumor-bearing hosts and the rationale for strategies for therapeutic vaccination and immunotherapeutic targeting of Treg cells with drugs and microRNA