Federico Caligaris-Cappio

Chronic lymphocytic leukemia B cells are endowed with the capacity to attract CD4+, CD40L+ T cells by producing CCL22.

The natural history of B‐chronic lymphocytic leukemia (CLL) is not entirely explained by intrinsic defects of the neoplastic cell, but is also favored by microenvironmental signals. As CLL cells retain the capacity to respond to CD40 ligand (CD40L) and as CD4+ T cells are always present in involved tissues, we asked whether malignant CLL cells might produce T cell‐attracting chemokines. We studied the chemokine expression of CD19+/CD5+ malignant B cells from peripheral blood (PB), lymph nodes (LN) or bone marrow (BM) of 32 patients and found a major difference. LN‐ and BM‐, but not PB‐derived cells, expressed a readily detectable reverse transcription‐PCR band for CCL22 and one for CCL17 of variable intensity. CD40 ligation of PB cells induced the mRNA expression of both CCL22 and CCL17. CCL22 was also released in the culture supernatants. These supernatants induced the migration of activated CD4+, CD40L+ T cells expressing the CCL22 receptor, CCR4. T cell migration was abrogated by anti‐CCL22 antibodies. Immunohistochemistry and cytofluorography studies revealed that a proportion of CD4+ T cells in CLL LN and BM expressed CD40L. Our data demonstrate that malignant CLL cells chemo‐attract CD4+ T cells that in turn induce a strong chemokine production by the leukemic clone, suggesting a vicious circle, leading to the progressive accumulation of the neoplastic cells.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Respiratory function in systemic lupus erythematosus: relation with activity and severity

OBJECTIVES To determine whether patients with primary Sjögrens syndrome (SS), diagnosed according to San Diego criteria, had improvement in their laboratory or clinical features during treatment with hydroxychloroquine (6-7 mg/kg/day) for at least two years. METHODS The study population included 50 consecutive patients with primary SS who were diagnosed according to San Diego criteria, and in whom hydroxychloroquine was suggested as treatment. This group included 10 patients who were early dropouts (side effects or desire not to take antimalarial drugs) and 40 patients who received drugs for at least two years (range 24-48 months). In a subset of SS patients, values for ESR (westergren) and quantitative immunoglobulins were available for comparison. Improvement with therapy was defined as: (a) > or = 20% improvement in variables of tear flow (Schirmers test I) or corneal integrity (rose Bengal): (b) > or = 20% salivary function (flow rate); and (c) improvement in at least two of the following measures: physicians assessment of global disease activity by > or = 20%, patient assessment of improvement in pain or fatigue by > or = 20%, and ESR improved by > or = 20 mm/hr. RESULTS In a retrospective study of SS patients who completed the trial, a significant improvement was noted in ocular symptoms (pain and dryness) in patients (55 and 57%) and improved corneal integrity (rose Bengal straining) in 53% of patients. The Schirmers test was improved by > or = 2 mm/5 minutes in 50% in patients. Improvement was noted in oral symptoms (pain and dryness) in patients (57 and 60%) and salivary flow rate was increased in 82% of patients. In a subset of SS patients evaluated, the ESR improved by > or = 20 mm/hr in 17/32 patients (53%) and quantitative IgG level by > or = 20% in 8/13 patients (61%). Physician global assessment of overall patient status and patient assessment of overall status indicated improvement in over 62% of patients. CONCLUSION In a retrospective study of patients fulfilling San Diego Criteria for SS, we found: (a) sustained improvement of local symptoms (painful eyes, painful mouth) and improvement of systemic manifestations (arthralgias and myalgias) after treatment with hydroxychloroquine 6-7 mg/kg/day over mean three-year follow-up; (b) laboratory analysis showed a significant improvement in their ESR and their quantitative IgG levels; (c) no significant late toxicity was observed in this study cohort. A prospective study of hydroxychloroquine in patients fulfilling San Diego criteria for SS is indicated.The objective of this study was to examine the relation between respiratory function tests, disease activity and disease severity in ambulatory patients with systemic lupus erythematosus (SLE) who did not present with overt respiratory problems. Lung volumes, maximal expiratory flows at 50% and 25% of vital capacity (MEF50 and MEF25), bronchial threshold to methacholine (PD15FEV 1), transfer factor CO (KCO) were measured in 24 consecutive SLE outpatients (22 women, age 41 ± 14.8 years) and in 24 healthy controls matched for age and sex. In SLE patients alveolar-arterial oxygen gradient (AaO2) was also measured. Disease activity was assessed by European Consensus Lupus Activity Measurement (ECLAM) scoring system and disease severity by Lupus Severity of Disease Index. In comparison to controls SLE patients showed a significant decrease of total lung capacity (TLC) (91.7 ± 16.5 vs 102.7 ± 12.9% predicted, P < 0.01), MEF25 (58.4 ± 25.2 vs 73.5 ± 19.5% predicted, P < 0.005), PD15FEV1 (2164 ± 1122 vs 4230 ± 1014 μg methacholine, P < 0.0001) and KCO (77.1 ± 20.5 vs 96.3 ± 12.4% predicted, P < 0.001). AaO2 (mean value 13.2 ± 8.4) was abnormally high (>20 mm Hg) in 12 patients. The ECLAM score of activity was inversely related with KCO (r = 0.48, P < 0.02). The severity index was significantly related with FEV1/VC ratio (r = 0.43, P < 0.05), MEF50 (r = 0.51, P < 0.01), MEF25 (r = 0.40, P < 0.05) and PD15FEV1 (r = 0.51, P < 0.01). In eight patients, evaluated also after treatment intensification, there was a significant increase in KCO (from 71.8 ± 24.7 to 84.9 ± 22.3% predicted, P < 0.01) along with a decrease in ECLAM score (from 3.0 ± 1.34 to 0.69 ± 0.75, P < 0.01). The relation between disease activity and KCO suggests a relation between systemic and alveolar inflammation whereas the relation between severity index, airway patency and reactivity indices suggests a cumulative damage to the airways in SLE patients, even in the absence of overt respiratory manifestations.

CD30 and type 2 T helper (Th2) responses.

CD30 is one of the members of the tumor necrosis factor receptor superfamily, originally described as a marker of Reed‐Sternberg and Hodgkins cells in Hodgkins lymphoma. CD30 appears to be preferentially expressed on, and its soluble form (sCD30) released by, CD4+ and CD8+ T cell clones capable of producing T helper 2 (Th2)‐type cytokines. In nonneoplastic conditions, CD30+ T cells are barely detectable in vivo; however, a few allergen‐specific CD4+CD30+ T cells inducible to the production of Th2‐type cytokines could be sorted out from the circulation of allergic subjects after allergen exposure. Moreover, high numbers of CD30+ T cells were found in the lymph node of a patient suffering from Omenns syndrome, a rare congenital Th2‐mediated immunodeficiency disorder. More importantly, high serum levels of sCD30 were observed in some conditions in which a pathogenetic role for Th2 cells has been suggested, such as Omenns syndrome, atopy, systemic lupus erythematosus, and after infection with measles virus or human immunodeficiency virus. Thus, detection of CD30+ T cells and/or of increased levels of sCD30 may reflect the presence of immune responses or immune alterations characterized by the prevalent activation of Th2‐like cells. J. Leukoc. Biol. 57: 726‐730; 1995.

Normal equivalent cells of B cell malignancies: analysis with monoclonal antibodies

Summary. Immunoglobulin heavy chain expression and reactivity of monoclonal antibodies RFA‐1, ‐2, ‐3, ‐4 and OKT10 discriminate between majority B lymphocyte populations in the bone marrow and in peripheral lymphoid organs. In this study normal tissues and various B cell malignancies were studied in cell suspensions and tissue sections. Pre‐B acute lymphoblastic leukaemia and multiple myeloma apparently reflect the phenotypes on normal B lineage cells of the bone marrow, while centroblastic‐centrocytic lymphoma, B chronic lymphoid leukaemia (CLL) and prolymphocytic leukaemia (PLL) show the characteristics of distinct peripheral B lymphoid subsets found at different sites in the lymphoid tissue. These ‘normal equivalent’ cells are centroblasts and centrocytes in the germinal centre, CLL‐like cells at the edge of the germinal centre (a minority population) and strongly Ig positive cells in the lymphocyte corona. Malignant cells in macroglobulinaemia are apparently more closely related to PLL and the corresponding normal peripheral B cells (in the corona) than to myeloma cells or the equivalent normal plasma cells in the bone marrow.

Cytokines involved in the progression of multiple myeloma.

We 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‐1β, IL‐3, IL‐4, IL‐6, IL‐7, IL‐8 and tumour necrosis factor‐alpha (TNF‐α) 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 IL‐1β, TNF‐α, 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 of the disease. Third, MM BM stromal cells are active IL‐6 and IL‐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.

In leukaemic CD5+ B cells the expression of BCL‐2 gene family is shifted toward protection from apoptosis

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl‐2 gene family. The pattern of expression of Bcl‐2, Bcl‐xL, Bcl‐xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B‐cell chronic lymphoid leukaemias.

Differential Effects on CLL Cell Survival Exerted by Different Microenvironmental Elements

Selected microenvironmental stimuli confer to leukemic cells a growth advantage and an extended survival. We aimed at dissecting the differential support provided by the different cellular components of the microenvironment where CLL cells accumulate. To this end we cultured purified CLL cells in vitro in the presence or absence of different accessory cells (stromal cells, autologous T lymphocytes) and/or soluble molecules (IL-4, sCD40L) and assessed the leukemic cell response in terms of cell viability and chemoattracting capacity. The results indicate that both T lymphocytes and stromal cells are involved in sustaining the survival of leukemic B cells, but indicate that their support is different in terms of time of onset and duration. T cells have a short-term support activity while stromal cells provide long-term support.

Immunohistochemical demonstration of follicular dendritic cells in bone marrow involvement of B‐cell chronic lymphocytic leukemia

Using a sensitive immunohistochemical technique and a specific monoclonal antibody (RFD‐3), follicular dendritic cells (FDC) have been demonstrated in a significant proportion (6/12) of bone marrow samples from patients with nodular marrow involvement of B‐cell chronic lymphocytic leukemia (B‐CLL). In all cases with “packed marrow” involvement and advanced stages of disease the FDC were absent. As these accessory cells are normally present only in the lymph nodes, their presence in the bone marrow is in accord with the view that B‐CLL might be the malignancy of an immature subpopulation of lymph node lymphocytes that invade the bone marrow.

Primary lymphoma of the heart. A case report and review of the literature

Primary cardiac lymphoma (PCL) is a rare and usually fatal neoplasm, which may cause syncope, arrhythmia, heart failure and pericardial effusion as presenting clinical complaints. A case of PCL in a 72-year-old man with moderate aortic stenosis is presented. The patient was investigated because of pericardial effusion and diagnosis of diffuse large B-cell lymphoma was obtained by open-chest biopsy of the heart. Fatal ventricular arrhythmia developed the day after the first course of chemotherapy. Clinical presentations and diagnostic approach of this rare tumour are discussed. While chemotherapy is the only effective treatment of PCL, early post-chemotherapy phase should be considered critical in patients with PCL, as suggested by other reported fatal complications in this period.