Loss of collagen type IV in rheumatoid synovia and cytokine effect on the collagen type-IV gene expression in fibroblast-like synoviocytes from rheumatoid arthritis.

Collagen type IV is a structural matrix protein which contributes to the structural organization of the synovia. In order to characterize the distribution of this protein in synovia with chronic synovitis, collagen type IV was detected by immunochemistry in normal synovia and in synovia from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). A decrease of collagen type IV was observed in synovial layers of rheumatoid synovia, which statistically correlated with the grade of inflammation and with the thickness of the synovial layer. In vitro, we found no differences in the gene expression of collagen type IV in cultures of fibroblast-like synoviocytes (FLS) derived from OA and RA using a reverse-transcriptase polymerase chain reaction. Nevertheless, we observed a downregulating effect of tumor necrosis factor-alpha and interleukin (IL)-1beta on the gene expression of collagen type IV only in FLS isolated from patients with RA. The effect of IL-1beta was dose dependent. In summary, we observed an inflammation-associated decrease of collagen type IV in the synovial layer of rheumatoid synovia. Inflammatory cytokines may play a role in regulating the synthesis of collagen type IV in the rheumatoid process in vivo.

Loss of laminin and of the laminin receptor integrin subunit alpha 6 in situ correlates with cytokine induced down regulation of alpha 6 on fibroblast-like synoviocytes from rheumatoid arthritis.

OBJECTIVE: To investigate in situ the expression of the integrin receptor subunits alpha 6 and beta 1 and the distribution of the ligand laminin in the synovia from osteoarthritis (OA) and rheumatoid arthritis (RA) patients and to study the effect of cytokines and antirheumatic drugs on the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of fibroblast-like synoviocytes (FBS) derived from OA and RA. METHODS: The expression of the alpha 6 and beta 1 integrin subunits and the distribution of laminin were examined immunohistochemically in normal synovia and in synovia from patients with OA and RA. The effect of proinflammatory cytokines (IL1 beta and TNF alpha), and of antirheumatic drugs (salicylic acid, dexamethasone, and methotrexate) on the alpha 6 and beta 1 expression of cultured normal FBS and FBS from patients with OA and RA was determined by flow cytometry. RESULTS: In normal synovia and in OA synovia samples with a low grade of inflammation, synovial lining cells (SLC) showed a parallel expression and distribution of alpha 6 and laminin. In synovia samples of OA with a higher grade of inflammation and in the majority of RA synovia samples laminin was pericellularly distributed in a low number of SLC, whereas alpha 6 was expressed on the surface of a high number of SLC. In RA synovia samples with severe inflammatory changes the gradual loss of laminin generally corresponded to a decrease of the alpha 6 integrin subunit. beta 1 was always strongly expressed in all synovia samples detected. Proinflammatory cytokines up regulated the expression of alpha 6 and beta 1 on OA-FBS, whereas these effectors decreases alpha 6 and beta 1 on RA-FBS. In contrast, antirheumatic drugs, in particular methotrexate and dexamethasone, reduced the expression of alpha 6 and beta 1 on OA-FBS, whereas the same treatment on RA-FBS stimulated the expression of these integrin subunits. CONCLUSION: The gradual loss of laminin in chronic synovitis may contribute to the altered expression of alpha 6 in SLC. IL1 beta and TNF alpha down regulated the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of FBS derived from RA. Therefore, these cytokines may be among the effectors regulating the expression of the alpha 6 integrin subunit in SLC in vivo. As antirheumatic drugs increase the expression of alpha 6 on RA-FBS, the presence of the laminin receptor may confer a protective effect on the synovia in vivo.

[Malignant lymphoma. Diagnosis and after-care in adults with special reference to cross-sectional imaging]. / Maligne Lymphome. Diagnostik und Nachsorge beim Erwachsenen unter besonderer Berücksichtigung der Schnittbildverfahren.

Radiological staging continues to remain the basis of a critical therapy of malignant lymphoma. As staging system, the Ann Arbor classification with some added modification is used. Up to now, CT is the imaging study of choice for staging and follow-up of cases of lymphoma. In future however, due to the advantages of the MRI, parts of the staging will be performed by MRI only. Imaging studies provide accurate measurement of extent of nodal disease. The detection of extra-nodal disease depends on the growth pattern and on the location. Focal lesions of sufficient size can be readily detected, diffuse infiltration is often missed. Computed tomography precisely reflects pathologic changes of lung involvement, but the findings are not specific. One weakness of the imaging studies is the low detection rate of splenic and hepatic involvement. Staging of gastrointestinal lymphoma has been improved by "hydro-CT" or "Hydro-MRI".

Differential expression and functional behaviour of the alpha v and beta 3 integrin subunits in cytokine stimulated fibroblast-like cells derived from synovial tissue of rheumatoid arthritis and osteoarthritis in vitro.

OBJECTIVE: The aim of this study was to investigate in situ the expression of the classic vitronectin (VN) receptor consisting of the alpha v and beta 3 subunits in synovial lining cells (SLC) of chronic synovitis occurring in osteoarthritis (OA) and in rheumatoid arthritis (RA). The expression and function of alpha v and beta 3 as VN receptor in cultured fibroblast-like synoviocytes (FBS) derived from patients with OA and RA was also compared. METHODS: Expression of alpha v and beta 3 was examined immunohistochemically in normal synovial tissue and in synovial tissue from patients with OA and RA. The effect of proinflammatory cytokines and of a synovial fluid of a patient with RA on the expression of the alpha v and beta 3 subunits of cultured FBS was determined by flow cytometry. Binding of OA and RA-FBS to VN was quantified using adhesion assays and the effect of interleukin 1 beta (IL1 beta) and tumour necrosis factor alpha (TNF alpha) on adhesion was measured. The specificity of the adhesion was tested by inhibition studies using monoclonal antibodies to integrin subunits. RESULTS: In in situ studies normal SLC showed a parallel distribution of alpha v and beta 3 subunits. OA-SLC strongly and uniformly expressed alpha v whereas RA-SLC showed heterogeneous expression of alpha v. In situ both OA-SLC and RA-SLC lacked the expression of the integrin subunit beta 3. In in vitro studies, OA-FBS and RA-FBS did not differ as regards expression of alpha v and beta 3, and VN attachment. Binding of RA-FBS to VN was partially blocked by antibodies against alpha v, beta 1, and beta 3 subunits, whereas only antibodies against alpha v and beta 3 inhibited the binding of OA-FBS to VN. The proinflammatory cytokines TNF alpha and IL1 beta increased the expression of alpha v and beta 3, and the VN binding of OA-FBS, whereas alpha v and beta 3 expression, and VN binding were downregulated in RA-FBS. Similar effects were found when the synovial fluid of an RA patient was used. CONCLUSION: The integrin subunit beta 3 seems to be one partner but not the major one with which the subunit alpha v forms functional vitronectin receptors in OA-FBS and RA-FBS. The interaction between synovial cells and inflammatory cytokines seems to be different for OA and RA; the basis for this difference, however, remains to be established.

[The significance of cardiolipin antibodies in systemic lupus erythematosus]. / Die Bedeutung von Cardiolipinantikörpern beim systemischen Lupus erythematodes.

The aim of the study was to investigate the frequency and clinical significance of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). 32 of 100 patients with SLE had positive anticardiolipin antibodies. Increased aCL were associated with thrombosis, thrombocytopenia, miscarriage, vasculitic skin changes and neurological symptoms. The incidence of thrombosis, thrombocytopenia, and neurological symptoms was significantly increased in the aCL-positive group as compared to the aCL-negative group. These findings confirm the results of former investigations and underline the role of aCL in systemic lupus erythematosus.

Regional adoptive immunotherapy with interleukin-2 and lymphokine-activated killer (LAK) cells for liver metastases.

A phase lb trial of a novel regional approach to adoptive immunotherapy is reported. Patients with liver metastases received continuous high-dose infusion of interleukin-2 (IL-2) into the splenic artery or intravenous infusion with subsequent transfer of lymphokine-activated killer (LAK) cells into the portal vein or the hepatic artery. Trafficking studies revealed homogeneous distribution of the LAK cells within the liver. The usual side-effects of IL-2 and LAK cells occurred without limiting liver toxicity. One partial (7+ months) and two complete responses (36 and 26+ months) were observed in 9 patients with metastases from cutaneous melanoma. None of 6 patients with metastases from ocular melanoma responded.

Interferon-alpha and interleukin-2 in the treatment of metastatic melanoma. Comparison of two phase II trials.

BACKGROUND: Interferon-alpha (IFN alpha) and interleukin-2 (IL-2) are active agents against malignant melanoma. There is, however, no consensus on the optimal dosing schedule of both drugs. This is a report of two sequential immunotherapy trials in patients with metastatic melanoma using two different IL-2 dosing schedules. METHODS: Schedule A consists of IFN alpha, 10 million U/m2/day subcutaneously for 5 days, followed by continuous intravenous infusion of IL-2, 1 mg/m2/24 hours for 5 days. Schedule B consists of the same dose of IFN alpha, but a modified regimen of IL-2. To improve the induction of high-affinity IL-2 receptors, the initial IL-2 dose was increased (1 mg/m2/6 hours, followed by 1 mg/m2/12 hours, and 1 mg/m2/24 hours). To reduce toxicity, the dose was reduced thereafter to 0.25 mg/m2/24 hours for the following 3 days. Both regimens were repeated after 4 weeks. RESULTS: 27 patients were treated with schedule A with a response rate of 18% (1 complete response [CR], 4 partial responses [PR]), 95% confidence interval, 6-36%. The response rate in 27 patients treated with schedule B was 41% (3 CR, 8 PR), 95% confidence interval, 22-61%. Severe, often dose-limiting toxicity was associated with IL-2 in schedule A, particularly hypotension and fluid retention. Toxicity was reduced significantly in schedule B. Maximal serum levels of soluble CD25 were 17,022 +/- 13,070 U/ml in schedule A, and 31,148 +/- 4227 U/ml in schedule B (P < or = 0.01). Serum levels of TNF alpha were significantly lower in schedule B than in schedule A, as were the side effects. CONCLUSIONS: Toxicity of IL-2 is reduced by modifying the schedule of administration, which also enhances the immunologic response and appears to increase the response rate.

Regional administration of lymphokine-activated killer cells can be superior to intravenous application.

A patient with liver metastases of human lymphocyte antigen (HLA) class II-negative malignant melanoma was treated with several cycles of adoptive immunotherapy with interleukin-2 and lymphokine-activated killer (LAK) cells. The authors evaluated the efficacy of regional transfer of LAK cells versus systemic intravenous administration. Initially, the patient was treated according to a regional treatment protocol, consisting of perfusion of the spleen with interleukin-2 and transfer of LAK cells into the portal vein; a partial remission was observed. Because of technical problems, interleukin-2 and LAK cells were administered intravenously in a second treatment cycle. This systemic treatment course resulted only in a minor mixed response of the hepatic metastases. A third treatment course was administered with the use of intravenous interleukin-2 infusion and arterial perfusion of the liver with LAK cells. The patient had separate hepatic arteries to both lobes of the liver as an anatomic variation. Because most of the tumor mass was present in the right lobe of the liver, a third of the LAK cells were injected into the right hepatic artery and the remaining cells were administered intravenously. The lesions in the right lobe of the liver regressed, but disease progression occurred in the left lobe. A fourth treatment cycle, consisting of intravenous infusion of interleukin-2 and arterial perfusion of both lobes of the liver with LAK cells, resulted in a complete response of all hepatic lesions, which has lasted 18 months to date. Because, in this patient, tumor regression was observed only in anatomic areas of the liver, which were perfused with LAK cells, it is suggested that the regional administration of LAK cells was essential for successful treatment.

Etoposide in acute leukemia. Past experience and future perspectives.

Etoposide as a single agent is active in relapsed and refractory acute myelogenous leukemia (AML), with complete responses (CR) rates of 10% to 25%. The drug has been safely combined with cytarabine, azacytidine, vinca alkaloids, and anthracyclines, inducing remission rates of 20% to 60% in patients with previously treated AML. The experience with etoposide in acute lymphoblastic leukemia is less extensive, but the drug seems to be active in combination with cytarabine or aclacinomycin. In addition, etoposide is combined with cytarabine and anthracyclines for the primary treatment of AML. The response rates thus achieved are comparable with those obtained with standard regimens. A Phase I/II trial was initiated to study the efficacy of the NOVE combination (mitoxantrone [10 mg/m2/d, days 1 to 5] plus etoposide [100 mg/m2/d for 3, 4, or 5 days] in patients with refractory AML. The results showed that extended duration of etoposide administration is associated with higher CR rates. Overall, a CR rate of 43% was achieved in 61 patients. A sequential regimen with IDAC (idarubicin/cytarabine) and NOVE was designed for primary treatment of adult patients with AML. Cycles of IDAC or NOVE are applied depending on response. The results of the pilot study with this strategy were encouraging with 18 of 20 patients achieving CR. Further studies are under way to verify the efficacy of this strategy.

Peripheral blood mononuclear cell collection from patients undergoing adoptive immunotherapy or peripheral blood-derived stem cell transplantation and from healthy donors.

The demand for collection of mononuclear cells from the peripheral blood of patients for therapeutic purposes is rapidly increasing. Automated blood cell separators are usually designed for collection of blood components from healthy donors. We reviewed safety and efficiency of collection data of a new procedure for the Fenwal CS 3000 blood cell separator in 125 collections from normal donors and 101 collections from patients after IL-2 pretreatment or chemotherapy. The new procedure set red blood cell spillovers to occur at 3.5 minute intervals, using procedure 1 with the interface detector set at 1,000 and the standard granulocyte and collection chambers. Despite significant anemia and thrombocytopenia in a large number of patients no serious procedure-related side effects occurred. The lymphocyte yield was 4.74 +/- 1.6 x 10(9) per 5 liters of blood processed in normal donors and 24.2 +/- 12.0 x 10(9) per 10 liters of blood processed after IL-2 treatment. After chemotherapy the lymphocyte yield was 4.5 +/- 3.1 x 10(9) per 10 liters of blood processed; the collection efficiency was found to be significantly lower in this group. The main problem was the platelet loss of 35.6 +/- 12% of the initial count in normal donors, 40.3 +/- 14.1% after IL-2 treatment, and 42.1 +/- 18.0% after chemotherapy. The platelet loss is, however, closely related to the preapheresis platelet count; patients with thrombocytopenia lose fewer platelets than normal donors. Therefore the procedure was found to be safe for patients with a platelet count as low as 20/nl. This report provides a basis for safe, effective mononuclear cell collection from patients with very abnormal peripheral blood counts.

A modified cytotoxicity assay with high sensitivity.

Since adoptive immunotherapy using lymphokine activated killer (LAK) cells and interleukin-2 (IL-2) has been introduced into clinical medicine, there has been a growing interest in cytotoxicity assays. The standard 51chromium release assay has certain disadvantages, in particular limited sensitivity, because of a high, nonspecific background release. We examined the conditions under which tritiated thymidine, which has been used to assess slow macrophage mediated cytolysis, can be utilized to assess the rapid cytotoxic activity of unstimulated PBL and LAK cells. The optimal assay duration for the 3H-thymidine (3H-TdR) release assay is 24 h. Under standard conditions actively proliferating effector cells do incorporate some of the 3H-TdR released by the target cells during this time, leading to false low results. This problem can be abolished by the addition of excessive amounts of cold TdR to the assay medium. We found a good correlation of the results of the TdR release assay and the Cr release assay. Using the very sensitive TdR release assay, unexpected significant cytolysis of the so-called 'NK resistant' Daudi cells by unstimulated PBL is demonstrated. The modified 3H-TdR release assay is well-suited to monitor the immunological effects of immunotherapy, using IL-2 and LAK cells.

The plasmacytoid T cell or plasmacytoid monocyte--a sessile lymphoid cell with unique immunophenotype and unknown function, still awaiting lineage affiliation.

Clusters of cells with a plasmacytoid appearance have been identified in T zones of human lymphoid tissue. These cells were shown to express the CD4 antigen in the absence of B-cell antigens. Thus, they were named "plasmacytoid T cells" (PTCs). Recent studies, however, have suggested a myelomonocytic origin of this cell type, which led to the designation "plasmacytoid monocytes." In order to obtain a comprehensive immunophenotype of this cell, we used frozen serial sections of lymph nodes, an indirect immunoperoxidase technique, and a broad panel of well-defined monoclonal antibodies (mAbs). On PTCs, the antigenic density was high for CD4 and CD26 antigens and low for CD2; they furthermore reacted with the mAb OKM5 recognizing an undefined antigen associated with the myelomonocytic lineage and expressed the monocyte-associated antigen CD68. PTCs expressed HLA-A, B, C, HLA-DR, and the HLA-D associated invariant chain (Ii) while HLA-DP antigens were expressed only in low amounts and HLA-DQ antigens were lacking entirely. PTCs further expressed the adhesion molecule CD11c. The absence of detectable transferrin receptor and the proliferation antigen defined by Ki-67 indicated that PTCs were nonproliferating. Other well-defined antigens confined to the T-, B-, and myelomonocyte lineages could not be detected in the target cell population. For us, antigenic equipment is still suggestive of a sessile, terminally differentiated T cell likely to exert a secretory function as yet unknown.