Gina Lama

B cell depletion in diffuse progressive systemic sclerosis: safety, skin score modification and IL-6 modulation in an up to thirty-six months follow-up open-label trial

IntroductionAn over-expression of CD19 has been shown in B cells of systemic sclerosis (SSc) and B cells are thought to contribute to the induction of skin fibrosis in the tight skin mouse model. The aim was to define the outcome on safety and the change in skin score after rituximab therapy in SSc patients and to correlate the clinical characteristics with the levels of interleukin (IL)-6 and with the immune cell infiltrate detected by immunohistochemistry.MethodsNine patients with SSc with mean age 40.9 ± 11.1 years were treated with anti-CD20, 1 g at time 0 and after 14 days. Skin biopsy was performed at baseline and during the follow-up. B-cell activating factor (BAFF) and IL-6 levels were also determined at the follow-up times.ResultsAfter 6 months patients presented a median decrease of the skin score of 43.3% (range 21.1-64.0%), and a decrease in disease activity index and disease severity index. IL-6 levels decreased permanently during the follow up. After treatment, a complete depletion of peripheral blood B cells was observed in all but 2 patients. Only 3 patients presented CD20 positive cells in the biopsy of the involved skin at baseline.ConclusionsAnti-CD20 treatment has been well tolerated and SSc patients experienced an improvement of the skin score and of clinical symptoms. The clear fall in IL-6 levels could contribute to the skin fibrosis improvement, while the presence of B cells in the skin seems to be irrelevant with respect to the outcome after B cell depletion.Trial registrationISRCTN77554566.

B cells in systemic sclerosis: A possible target for therapy

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition in the skin and other visceral organs and it is associated with immune activation characterized by autoantibody production, release of various cytokines and T-lymphocyte activation. Several recent lines of evidence in animal models and in SSc patients indicate a potential role for B cells in the SSc. B cells have arisen as a possible player in tissue fibrosis in some experimental models and, since IL-6 produced by B cells, along with TGF-β, may induce matrix synthesis and less collagen degradation, targeting B cells could be one way to reduce ECM deposition and reduce the inflammatory background. Both SSc patients and tight-skin mice, a genetic model of SSc, have intrinsic B-cell abnormalities characterized by chronic B-cell activation. SSc patients present an increased number of naïve B cells and an activation of memory B cells, despite a reduction in their number. B cells from SSc patients exhibit increased expression of CD19. Remarkably, CD19 loss or B-cell depletion using antimouse CD20 antibody suppresses the development of skin hyperplasia and autoimmunity in tight-skin mice. Additionally, recent studies revealed a possible beneficial effect of anti-human CD20 antibody (Rituximab) therapy on skin fibrosis and lung involvement in SSc patients. These studies reported also the safety of Rituximab in SSc patients. All these findings suggest a possible role of antiCD20 treatment in SSc patients.

Invasive tumor cells and prognosis in a selected population of patients with glioblastoma multiforme

After surgical resection, the residual, invasive glioblastoma (GBM) cells give rise to a recurrent tumor, which, in 96% of patients, arises adjacent to the resection margin.

Sensitivity of human melanoma cells to oestrogens, tamoxifen and quercetin: is there any relationship with type I and Ii oestrogen binding site expression?

We investigated the effect of oestrogens, anti-oestrogens and flavonoids on the growth of a human melanoma cell line (SK-Mel-28) and, at the same time, the presence of both type I oestrogen receptors (ERs) and type II oestrogen binding sites (type II EBS) to gain a fuller picture of the relationship between melanoma cell proliferation and receptor status. 17β-Oestradiol (E2) and the flavonoid quercetin (Q) produced a marked inhibition of proliferation, but only at the highest dose used (10-5 M) and only when added daily to the medium. Diethylstilboestrol (DES) (10-5M) was effective in inhibiting cell growth when the medium was renewed every 3 days and produced a more pronounced reduction when added daily to the medium. Tamoxifen (TAM) inhibited cell proliferation at a dose starting from 10-7 M when the medium was renewed every 3 days. When added daily to the medium, it did not induce a greater inhibitory effect and it was cytotoxic at 5 x 10-6 M and 10-5 M. The antiproliferative effect of E2, DES and Q did not seem to be dependent on their interaction with ERs, which were minimally detected in SK-Mel-28 in both immunocytochemical and biochemical assays. Our model revealed, through a biochemical assay, a large number of type II EBSs which could be involved in the anti-oestrogen action, but this does not exclude the involvement of other mechanisms. Finally, TAM (10-5 M) appeared to reduce the activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, an effect that could be interesting from the point of view of the therapeutic efficacy of alkylating agents.

The impact of repeated surgery and adjuvant therapy on survival for patients with recurrent glioblastoma

OBJECTIVE Treatment of glioblastoma recurrence can have a palliative aim, after considering risks and potential benefits. The aim of this study is to verify the impact of surgery and of palliative adjuvant treatments on survival after recurrence. METHODS From January 2002 to June 2008, we treated 76 consecutive patients with recurrent glioblastoma. Treatment was: 1-surgery alone--17 patients; 2-adjuvant-therapy alone--24 patients; 3-surgery and adjuvant therapy--16 patients; no treatment--19 patients. The impact on median overall-survival (OS-time between recurrence and death/last follow-up) of age, Karnofsky performance scale (KPS), resection extent and adjuvant treatment scheme (Temozolomide alone vs low-dose fractionated radiotherapy vs others) was determined. Survival curves were obtained through the Kaplan-Meier method. Cox proportional-hazards was used for multivariate analyses. Significance was set at p<0.05. RESULTS Median OS was 7 months. At univariate analysis, patients with a KPS≥70 had a longer OS (9 months vs 5 months--p<0.0001). OS was 6 months for patients treated with surgery alone, 5 months for patients that received no treatment, 8 months for patients treated with chemotherapy alone, 14 months for patients treated with surgery and adjuvant therapy--p=0.01. Patients with a KPS<70 were significantly at risk for death - HR 2.8 - p=0.001. Subgroup analysis showed no significant differences between patients receiving gross total or partial tumor resection and among patients receiving different adjuvant therapy schemes. Major surgical morbidity at tumor recurrence occurred in 16 out of 33 patients (48%). CONCLUSION It is fundamental, before deciding to operate patients for recurrence, to carefully consider the impact of surgical morbidity on outcome.

Progenitor/Stem Cell Markers in Brain Adjacent to Glioblastoma: GD3 Ganglioside and NG2 Proteoglycan Expression

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with &agr;-smooth muscle actin (&agr;-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Direct effects of GnRH agonists in human hormone-sensitive endometrial cells

The antiproliferative effect of two GnRH agonists (leuprorelin acetate and triptorelin), alone or combined with tamoxifen (TAM) or medroxyprogesterone acetate (MPA), on human estrogen-sensitive endometrial cancer cells (Ishikawa) was investigated. Although ineffective when tested alone in all the culture conditions used, both analogues counteracted or even suppressed the estrogen-stimulated growth of Ishikawa cells. The antiestrogenic effect of TAM or MPA was not modified by their association with high doses of the GnRH analogues, but low concentrations of triptorelin combined with MPA 10(-7) M determined a reduction in cell numbers which was greater than that obtained with the progestin or the analogue alone. In addition, analogue treatment prevented the estrogen-induced decrease in the level of estrogen receptors. Our data provide evidence that GnRH agonists can directly inhibit estrogen-stimulated endometrial cancer cell growth and suggest that they may interfere with steroid-receptor machinery.

Steroid receptor enhancement by natural interferon-β in advanced breast cancer

Abstract In the current study we investigated the effect of two different doses of natural interferon-beta (IFN-β) on steroid hormone receptors in 45 patients with advanced breast cancer. IFN-β seems to regulate the receptor mechanisms, inducing in cutaneous metastases an increase of oestrogen and progesterone receptors. Moreover, using IFN-β and tamoxifen as a combined therapy in 23 receptor-positive patients, no negative interference of the two drugs was observed and no relevant side-effects due to the treatment were noticed. The moduation of steroid receptor content by IFN-β in advanced breast cancer might represent an interesting way to ameliorate the clinical responsiveness to anti-oestrogens.

Leuprorelin Acetate Long-Lasting Effects on GnRH Receptors of Prostate Cancer Cells: An Atomic Force Microscopy Study of Agonist/Receptor Interaction

High cell-surface GnRH receptor (GnRH-R) levels have been shown to have a major influence on the extent of GnRH agonist-mediated tumor growth inhibition. The ability of the GnRH agonist leuprorelin acetate (LA) to induce a post-transcriptional upregulation of GnRH-R at the plasma membrane of androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer (PCa) cells has been previously demonstrated by Western blotting. Here we performed single molecule force spectroscopy by using Atomic Force Microscopy (AFM), which has proven to be a powerful tool allowing for investigation of living cell surface biological features, such as the so far unclear GnRH agonist/receptor interaction. Thus, in the hormone-insensitive PC-3 cells, we characterized the strength of the LA-receptor binding, and the amount and distribution of the functional receptor molecules on the cell surface. The effect of a long and continuous treatment (up to 30 days) with the agonist (10−11 and 10−6 M) on the same parameters was also investigated. A GnRH-R increase was observed, reaching the maximum (∼80%) after 30 days of treatment with the highest dose of LA (10−6 M). The analogue-induced increase in GnRH-R was also demonstrated by Western blotting. In addition, two different receptor bound strengths were detected by AFM, which suggests the existence of two GnRH-R classes. A homogeneous distribution of the unbinding events has been found on untreated and treated PC-3 cell surfaces. The persistence of high receptor levels at the membrane of these living cells may warrant the maintenance of the response to LA also in androgen-unresponsive PCa. Moreover, the determination of ligand/receptor bond strength could shed light on the poorly understood event of LA/GnRH-R interaction and/or address structural/chemical agonist optimizations.

Effects of natural beta-interferon and recombinant alpha-2B-interferon on proliferation, glucocorticoid receptor content, and antigen expression in cultured HL-60 cells

In the current study, we investigated the effects of natural beta‐interferon (β‐IFN) and recombinant alpha‐2b‐interferon (α‐IFN) on the growth of the HL‐60 cell line. Cells cultured in a medium that contains various concentrations (from 10 to 1000 IU/ml) of interferons showed a growth inhibition, which reaches the maximum after a 6‐day treatment, at the highest dose used. Furthermore, we studied the effect of both β‐IFN and α‐IFN on the level of glucocorticoid receptors. This was enhanced more than 30% with respect to control in HL‐60 cells exposed for 24 hours to concentrations of β‐IFN that ranged from 100 to 1000 IU/ml. The increase of the receptor amount was seen even if cells were treated for 5 days, and was not accompanied by a modification of antigen expression of HL‐60 cells. α‐IFN did not modify the glucocorticoid receptor level substantially in our experimental conditions. Our data indicate that both β‐IFN and α‐IFN regulate HL‐60 cell proliferation. Additional studies are required to clarify if modifications of the receptor level induced by β‐IFN could be related to the modulation of hormone‐sensitivity in this model.