Modulation of osteoclast-activating factor activity of multiple myeloma bone marrow cells by different interleukin-1 inhibitors.

We have studied the effects of several interleukin-1 (IL-1) inhibitors--IL-1 receptor antagonist (IL-1ra), soluble IL-1 receptor (sIL-1R) types I and II, and neutralizing monoclonal antibody (mAb) specific for IL-1 receptor type I--on the osteoclast-activating factor (OAF) activity of recombinant IL-1beta and of culture supernatants of unfractionated bone marrow mononuclear cells from multiple myeloma (MM) patients. The latter activity sharply correlated with the IL-1 content of culture supernatants (r = 0.949; p < 0.001). IL-1ra and sIL-1R types I and II had a clear-cut modulating effect on the OAF activity of IL-1beta at saturating doses (2-10 ng/mL); their effect was evident at 2 ng/mL and was dose-dependent over a large range of concentrations. Similarly, the three reagents neutralized the OAF activities of all MM cell supernatants in a dose-dependent fashion and completely abolished them when tested at the fixed concentration of 5 nM. The bone-resorbing activity of tumor necrosis factor-alpha (TNF-alpha) or lymphotoxin (LT), tested alone or added to MM cell supernatants, was affected not at all by IL-1ra and only minimally by sIL-1R types I and II, suggesting that little or no endogenous IL-1 was produced by the rat cells in the assay under TNF-alpha or LT stimulation. Consistent with these findings, PGE2 production elicited by IL-1beta or IL-1-rich supernatants in the rat long-bone assay was abolished by each reagent. Also, mAbs to the IL-1R p80 (type I) chains could modulate the effects of IL-1--recombinant or plasma cell-derived--in the OAF assay, but their activity was markedly less pronounced when compared with the IL-1 inhibitors, since they could never completely abolish bone resorption. Taken together, these findings demonstrate that inhibition of IL-1 interaction with cognate surface receptors on bone cells effectively counteracts its biologic activity. The findings also strongly indicate that OAF activity in conditioned medium of unfractionated myeloma bone marrow cells is predominantly, if not solely, related to IL-1beta.

Potential role of interleukin-1 as the trigger for diffuse intravascular coagulation in acute nonlymphoblastic leukemia.

Abnormalities of coagulation are common in patients with acute nonlymphoblastic leukemia, although the mechanisms involved are unclear, except in a few cases. To investigate the pathogenesis of this coagulopathy, suspensions of purified leukemic cells were prepared and tested for procoagulant activity. Neither the leukemic cells nor their supernatants directly accelerated the clotting of plasma. Since the leukemic cells did not possess direct procoagulant activity, their ability or inability to elaborate a mediator of cellular coagulant properties, interleukin-1, was studied. Leukemic cells from patients with coagulopathy elaborated interleukin-1, and addition of phytohemagglutinin increased interleukin-1 release. In contrast, no interleukin-1 was released, before or after stimulation with phytohemagglutinin, from leukemic cells from patients without coagulopathy. Leukemic cells from another group of patients with abnormalities of coagulation released interleukin-1 only after phytohemagglutinin treatment. In terms of the coagulation mechanism, interleukin-1 containing supernatants from leukemic cell cultures induced the procoagulant receptor tissue factor, a co-factor in the initiation of coagulation, on the endothelial cell surface. There was coordinate suppression of the anticoagulant endothelial cell receptor thrombomodulin, a co-factor for the antithrombotic protein C pathway. Antibody to interleukin-1 prevented these changes in cellular coagulant properties. Taken together, these changes result in a shift in the balance of endothelial cell coagulant properties to an activated state in which mechanisms promoting procoagulant reactions on the vessel surface predominate. Synthesis and release of the mediator interleukin-1 by leukemic cells thus defines a new mechanism through which malignant cells can potentially activate the coagulation mechanism.

Smoking cessation at the workplace. Results of a randomised controlled intervention study. Worksite physicians from the AIREL group.

OBJECTIVES: To compare the effects of a worksite intervention by the occupational physician offering simple advice of smoking cessation with a more active strategy of advice including a "quit date" and extra support. POPULATION: Employees of an electrical and gas company seen at the annual visit by their occupational physicians. CRITERIA END POINTS: Smoking point prevalence defined as the percentage of smokers who were non-smokers at one year. Secondary criteria were the percentage of smokers who stopped smoking for more than six months and the difference in prevalence of smoking in both groups. METHODS: Randomised controlled trial. The unit of randomisation was the work site physician and a random sample of the employees of whom he or she was in charge. The length of the follow up was one year. Each of 30 work site physicians included in the study 100 to 150 employees. RESULTS: Among 504 subjects classified as smokers at baseline receiving simple advice (group A) and 591 the more active programme (group B), 68 (13.5%) in group A and 109 (18. 4%) were non-smokers one year later (p=0.03; p=0.01 taking the occupational physician as the statistical unit and using a non-parametric test). Twenty three subjects (4.6%) in group A and 36 (6.1%) in group B (p=0.26) declared abstinence of six months or more. Among non-smokers at baseline, 3.4% in both groups were smokers after one year follow up. The prevalence of smokers did not differ significantly at baseline (32.9% and 32.4%, p=0.75). After the intervention the prevalence of smoking was 30.8% in group A and 28. 7% in group B (p=0.19). An increase of the mean symptoms score for depression in those who quit was observed during this period. CONCLUSIONS: A simple cessation intervention strategy during a mandatory annual examination, targeting a population of smokers independently of their motivation to stop smoking or their health status, showed a 36% relative increase of the proportion of smokers who quit smoking as compared with what can be achieved through simple advice.

[The nature of crystals in plasma cells present in a case of apparently benign biclonal gammapathy]. / Sulla natura dei cristalli endoplasmacellulari presenti in un caso di gammapatia biclonale apparentemente benigna.

A 77 years old white man presents, in his blood serum, a moderate gammapathy with 2 monoclonal peaks, IgAk and IgGlambda, and in his urine a little quantity of BJk, without any clinical or radiological manifestation. The sternal bone marrow showed 3-5% plasmacells, mostly containing many needlelike inclusions in the cytoplasm. These inclusions, red-violet at the May-Grünwald-Giemsa, were PAS and Sudan negative; positive at the Danielli reaction (for proteins). The alpha-naphtil-acetate-esterase and the acid phosphatase were present in one outer layer of the inclusions; ATP-ase was absent. At the electron microscopy, the inclusions were localized outside the rough endoplasmic reticulum; they exhibited a crystalline structure and were surrounded by an envelope which reminded the lysosomes. Basing on the morphological pattern, as well as on the presence of some lysosomal enzymes and on the lack of staining of crystals with fluorescinated anti-Ig sera, the hypothesis is stressed of an abnormal lysosomal hyperactivity, possibly leading to crystallization of the enzymatic proteins in the lysosomes.

AL-amyloidosis in monoclonal gammopathies.

Sixty-two patients affected by MGUS underwent fat tissue aspirate examination for diagnosis of AL amyloidosis. Nine out of the 62 were found to be Congo red positive. MGUS had already been diagnosed for quite a long time in about 60% of these patients, while this prevalence decreased to 24% among the Congo red negative patients. The follow-up of the positive patients is reported.

Circadian patterns in histamine concentrations and mast cell number in the rat thyroid gland.

We carried out a cross-sectional chronobiological investigation on blood histamine, thyroid histamine and thyroid mast cell number in Wistar rats. Daily, blood histamine varied from 0.38 +/- 0.01 (12.00 h) to 0.60 +/- 0.01 mg/g wet weight (20.00 h) and thyroid histamine from 21.2 +/- 1.19 (04.00 h) to 38.3 +/- 1.54 mg/g wet weight (08.00 h). The number of mast cells per microscopic field ranged from 10.8 +/- 0.6 (16.00 h) to 14.6 +/- 0.6 (12.00 h) in males and from 8.3 +/- 0.2 (04.00 h) to 14 +/- 0.4 (12.00 h) in females. Chronobiologic analysis indicates that the levels of all three variables follow a circadian pattern with a period of 12 h. Peak levels were noted at 07.36 h and 19.36 h for the blood histamine concentration, at 09.00 and 21.00 h for the tissue histamine concentration, and at 11.00 and 23.00 h for the mast cell number. The consistent, consecutive relationship of these data supports the hypothesis that thyroid mast cell number is conditioned by the blood histamine level and thyroid histamine concentration.

Cytochemical detection of histamine in the human granulopoietic series of healthy subjects and of patients affected by chronic myeloid leukaemia. A spectrophotofluorimetric test for checking OPT-induced fluorescence in isolated cells.

Cellular histamine in blood and bone marrow has been identified histochemically using an o-phthaldialdehyde fluorescence reaction. The specificity of the reaction was tested by a spectrofluorometric analysis of cell extracts. In normal blood, the basophils emit a bright yellow fluorescence, whereas neutrophils, eosinophils and platelets react less consistently and when they do, they give off a less intense yellow or blue emission. In normal marrow, basophils react strongly whereas the metamyelocytes and later granular cells show only a weak yellow or blue fluorescence. In chronic myeloid leukaemia, cells of the granular series emit a strong yellow fluorescence at all stages of development, although still less intense than the basophils. During remission, the fluorescence pattern of cells from leukaemic subjects reverts to that of normal cells.

[Glycosaminoglycans in the spleen of normal humans and in the spleen of subjects with chronic myeloid leukemia]. / Les glycosaminoglycanes dans la rate humaine normale et dans la rate de sujets atteints de leucémie myéloïde chronique.

The average glycosaminoglycan content in control spleens, expressed as uronic acid, was 0.23 +/- 0.02 mg/g of dry wt; the average glycosaminoglycans content in spleens of CML patients, expressed as uronic acid, was 0.91 +/- 0.23 mg/g of dry wt. In control and in leukemic spleens the same glycosaminoglycans were present, that is hyaluronic acid, heparan sulphate, dermatan sulphate, chondroitin-4-sulphate and chondroitin-6-sulphate. However, in leukemic spleens the normal quantitative relationship between these glycosaminoglycans was greatly modified; in fact in control spleens hyaluronic acid, heparan sulphate and the chondroitin sulphates were present in almost equal proportions, whereas in leukemic spleens the chondroitin sulphate group alone represented almost 9/10 of all the glycosaminoglycans. Since this proportion is weakly modified in leukemic spleens in which the number of myeloid cells has been notably reduced after chemotherapy, we may suppose that this phenomenon is due to the very marked modifications which take place in the micro-environment of the leukemic spleen.

Histamine uptake by human normal neutrophils in vitro.

The uptake of different concentrations of (2.5-3H) histamine by normal human neutrophils at 37.5 degrees C in a tris-albumin buffer was determined at 20, 60 and 90 min of incubation. Uptake was correlated with the concentration of amine and incubation time with a saturation-like curve. On the basis of these results, it is suggested that neutrophils play a role in the regulation of histamine plasma levels.

[Cytochemical demonstration and characterization of acid mucopolysaccharides in normal granulocytic series]. / Démonstration et caractérisation cytochimique des mucopolysaccharides acides des séries granulocytaires normales

Metachromatic granulations are seen in myeloblasts, promyelocytes, myelocytes, metamyelocytes and young mature cells, after staining by all metachromatic dyes, except methylene blue. Furthermore by screening at various pHs granular metachromasia is detectable starting at pH 3.2 in 6% of case, at pH 4 in 78% of cases, at pH 4.5 in 16% of cases. The use of toluidine blue 10(-4) M in solutions at varying pH, after methylation and after alkali demethylation demonstrate the acid sulfated mucopolysaccharide react within the pH range (3.2), 4, 4.5, 5; persistence of non-alcohol resistant granular metachromasia above pH 5.5 suggests the presence of granules containing acid mucopolysaccharides staining as the result of possessing --COOH radicals. This data was verified by other histochemical techniques (Alcian blue and PAS staining) which have shown positive granules. All the data on the metachromatic, alcian blue and PAS positive granulations, have been carefully confirmed by chemical and enzymatic reactions. The results of this study led us to the conclusion that azurophilic granulations are made of a sulfated polysaccharide of chondroitinsulfate group. In the more immature cells alcian blue positive granules (mucopolysaccharide of acid hyaluronic type) and PAS positive granules (polysaccharides with a low sulfation level) can be demonstrated. From this data, it is suggested that azurophilic granulations are the final event of an evolutive process of polysaccharide material, fundamentally based on a progressive sulfation.

Histamine uptake by leukocytes in vitro.

Human leukocytes at the concentration of 10(6) cells/ml were incubated for 1h at 37.5 degrees C in a tris-albumin buffer, containing 0, 6.25, 12.5, 25, 50, and 100 micrograms/ml of histamine. Histamine uptake was measured by a photofluorometric method. We found that the leukocytes take up exogenous histamine and attain respective intracellular concentration of 307%, 423%, 438%, 1000% and 645% above controls. It was found that neutrophils and eosinophils possess the greatest capacity to take up exogenous histamine whereas, contrary to the expectation, basophils display poor capacity.

Daily rhythmic variations in histamine in human blood.

We examined whether or not normal subjects have rhythmic changes of blood histamine levels. Daily predictable variations are present with 3 maxima and 3 minima and acrophase at 09.13. The significance of these changes is presently unknown.

[Cytochemical studies of bone marrow plasma cells in monoclonal gammapathies (author's transl)]. / Etude cyto-enzymologique des plasmocytes médullaires des dysglobulinémies monoclonales myélomateuses et non myélomateuses.

Three plasma cell enzymatic reactions--ATPase, acid phosphatase (AP), alpha-naphthyl acetate esterase (alpha-NAE)--were tested in a large number of bone marrow samples from patients with multiple myeloma (MM), benign monoclonal gammapathy (BMG)--idiopathic (iBMG) and secondary (sBMG)--, polyclonal hypergammaglobulinemia (PH), Waldenström's macroglobulinemia (WM) and from normoglobulinemic subjects (NS). The purpose of the present study was to evaluate the significance of these reactions in differential diagnosis of BMG and initial or atypical MM. For each reaction results were expressed as scores and normal limits were statistically established. Our findings in MM, HP and NS are consistent with previous reports (similar enzymatic pattern in NS and PH; depressed ATPase activity, raised AP and alpha-NAE activities in most MM cases). In the BMG class, ATPase activity was normal in about 40% of iBMG, while it was depressed in other cases; most sBMG had diminished ATPase activity. AP patterns were normal for both BMG groups. alpha-NAE activity was normal in 50% of iBMG, while it was raised in the other cases. All the reactions were normal in WM. In a bayesian analysis of cytochemical patterns of NS and MM subjects ATPase showed the highest diagnostic significance, followed by alpha-NAE and AP. ATPase, therefore, seems to be the most useful criterion for the diagnosis of initial MM; low ATPase score BMG patients should therefore be carefully followed-up.

Recurrent oral condylomata acuminata and hairy leukoplakia: an early sign of myelodysplastic syndrome in an HIV-seronegative patient.

Oral hairy leukoplakia (OHL) has been observed in all risk groups seropositive for HIV infection. Recently, this lesion has also been described in HIV-seronegative patients with immunosuppression of iatrogenic origin. We report on a HIV-1 and HIV-2 seronegative, heterosexual man affected by refractory anemia with ringed sideroblasts (myelodysplastic syndrome), who developed recurrent oral condylomata acuminata and OHL as an early clinical manifestation. The diagnosis of OHL was confirmed by identifying Epstein-Barr viral particles by electron microscopy and by in situ DNA hybridization. HIV infection was ruled out using polymerase chain reaction and testing for HIV-1 and HIV-2 antibodies.

Production of interleukin-1 by bone marrow myeloma cells.

Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT-specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti-IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.