We have verified the hypothesis that multiple myeloma (MM) may be disseminated by circulating clonogenic cells that selectively home to the bone marrow (BM) to receive the signal(s) leading to proliferation, terminal differentiation, and production of the osteoclast activating factors. Long-term cultures of stromal cells have been developed from the BM of nine patients with MM. These cells were mostly fibroblast- like elements, interspersed with a proportion of scattered macrophages and rare osteoclasts. BM stromal cells were CD54+, produced high levels of interleukin-6 (IL-6) and measurable amounts of IL-1 beta, and were used as feeder layers for autologous peripheral blood mononuclear cells (PBMC). After 3 weeks of cocultures, monoclonal B lymphocytes and plasma cells, derived from PBMC, developed and the number of osteoclasts significantly increased. Both populations grew tightly adherent to the stromal cell layer and their expansion was matched by a sharp increase of IL-6 and by the appearance of IL-3 in the culture supernatant. These data attribute to BM stromal cells a critical role in supporting the growth of B lymphocytes, plasma cells, and osteoclasts and the in vivo dissemination of MM.

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SUMMARYWe have investigated which of the cytokines that are relevant in the in vitro growth of multiple myeloma (MM) malignant plasma cells are actually produced in vivo by MM patients. To this end, we have measured the levels of IL-1^, IL-3, IL-4, IL-6, IL-7, IL-8 and tumour necrosis factor-alpha (TNF-a) both in sera and in the supernatant of bone marrow (BM) stromal cell cultures from patients with MM and monoclonal gammopathy of undetermined significance (MGUS). The significance of our findings is three-fold. First, IL-6 and IL-8 are produced by MM BM stromal cells, while \h-\ji, TNF-a, IL-4 and IL-7 are not. Second, IL-3 is the only cytokine consistently raised in serum samples; we have also detected low levels of serum IL-6 in a minority of cases, usually in advanced stage ofthe disease. Third, MM BM stromal cells are active IL-6 and lL-8 producers, while both normal and MGUS BM stromal cells are low producers, thus suggesting that in the BM of MM a number of environmental cells, that would normally be quiescent, are instead activated and that, in MM, activated BM stromal cells play an active role in supporting the progressive expansion of the B cell clone.

The BM microenvironment in MM, in terms of adhesive features, is well organized to entrap circulating precursors with BM-seeking properties and is able to produce cytokines that offer them the optimal conditions for local growth and final differentiation. Likewise, the malignant B cell clone is equipped with adhesion molecules which enable the cell to establish close contacts with BM stromal cells. Furthermore a number of cytokines are released including IL-1 beta and M-CSF activating BM stromal cells to produce other cytokines, such as IL-6, that stimulate the proliferation of plasma cells. Finally, most cytokines produced locally, including IL-1 beta, TNF-beta, M-CSF, IL-3 and IL-6, also have OAF properties, explaining why the expansion of the B cell clone parallels the activation and numerical increase of the osteoclast population.

CD5+ B‐chronic lymphocytic leukaemia (B‐CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl‐2 family of genes. Fludarabine (9‐β‐D‐arabinofuranosyl‐2‐fluoradenine, F‐ara‐A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F‐ara‐A‐induced apoptosis might be related to Bcl‐2 modifications and to evaluate in vitro/in vivo correlations.  Peripheral blood lymphocytes from eight B‐CLL and four leukaemic MCL were cultured in the presence of different concentrations of F‐ara‐A ±methylprednisolone (MP). F‐ara‐A down‐regulated the expression of Bcl‐2 in 5/12 cases. mRNA down‐regulation was maximal at 48 h; protein down‐regulation was prominent after 48 h. Both events were dose‐dependent. The amount of apoptosis was significantly higher in the samples treated with F‐ara‐A than in those exposed to MP alone. In the seven remaining cases, no Bcl‐2 down‐regulation was observed after exposure to F‐ara‐A and the degree of F‐ara‐A‐induced apoptosis overlapped that induced by MP.  The in vivo outcome after treatment with three to six courses of F‐ara‐A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl‐2 down‐regulation and prominent apoptosis after exposure to F‐ara‐A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl‐2 modulation after exposure to F‐ara‐A, two had a PR, but the other three did not show any in vivo clinical response.

Background The efficacy of early treatment with convalescent plasma in patients with COVID-19 is debated. Nothing is known about the potential effect of other plasma components other than anti-SARS-CoV-2 antibodies. Methods To determine whether convalescent or standard plasma would improve outcomes for adults in early phase of Covid19 respiratory impairment we designed this randomized, three-arms, clinical trial (PLACO COVID) blinded on interventional arms that was conducted from June 2020 to August 2021. It was a multicentric trial at 19 Italian hospitals. We enrolled 180 hospitalized adult patients with COVID-19 pneumonia within 5 days from the onset of respiratory distress. Patients were randomly assigned in a 1:1:1 ratio to standard of care (n = 60) or standard of care + three units of standard plasma (n = 60) or standard of care + three units of high-titre convalescent plasma (n = 60) administered on days 1, 3, 5 after randomization. Primary outcome was 30-days mortality. Secondary outcomes were: incidence of mechanical ventilation or death at day 30, 6-month mortality, proportion of days with mechanical ventilation on total length of hospital stay, IgG anti-SARS-CoV-2 seroconversion, viral clearance from plasma and respiratory tract samples, and variations in Sequential Organ Failure Assessment score. The trial was analysed according to the intention-to-treat principle. Results 180 patients (133/180 [73.9%] males, mean age 66.6 years [IQR 57–73]) were enrolled a median of 8 days from onset of symptoms. At enrollment, 88.9% of patients showed moderate/severe respiratory failure. 30-days mortality was 20% in Control arm, 23% in Convalescent (risk ratio [RR] 1.13; 95% confidence interval [CI], 0.61–2.13, P = 0.694) and 25% in Standard plasma (RR 1.23; 95%CI, 0.63–2.37, P = 0.544). Time to viral clearance from respiratory tract was 21 days for Convalescent, 28 for Standard plasma and 23 in Control arm but differences were not statistically significant. No differences for other secondary endpoints were seen in the three arms. Serious adverse events were reported in 1.7%, 3.3% and 5% of patients in Control, Standard and Convalescent plasma arms respectively. Conclusions Neither high-titer Convalescent nor Standard plasma improve outcomes of COVID-19 patients with acute respiratory failure. Trial Registration Clinicaltrials.gov Identifier: NCT04428021. First posted: 11/06/2020

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