IL-1beta expression in IgM monoclonal gammopathy and its relationship to multiple myeloma.

We have shown that IL-1beta is not detectable in normal plasma cells but is produced by plasma cells from virtually all patients with multiple myeloma (MM). To extend our earlier work, IL-1beta expression was determined in 13 newly diagnosed patients with IgM monoclonal gammopathy. Eleven patients with Waldenstrom's macroglobulinemia (WM) and two patients with IgM MM were investigated for IL-1beta expression by in situ hybridization (ISH). All patients with WM had bone marrow biopsies consistent with the diagnosis, an IgM M-protein in the serum, and subsequently required chemotherapy. Seven of 11 patients with WM had an M-protein >3 g/dl and five patients had bone surveys performed that were negative for osteolytic disease. Two patients were diagnosed with IgM MM because of the presence of significant osteolytic disease on a metastatic bone survey. ISH for kappa, lambda, and IL-1beta expression was performed on bone marrow aspirates from each of the 13 patients. None of the neoplastic cells from the 11 patients with WM showed detectable IL-1beta expression by ISH. However, the neoplastic cells from both patients with IgM MM expressed IL-1beta mRNA at high levels. This aberrant IL-1beta production may explain the presence of bone lesions in the patients with IgM MM.

Knowledge about genetic risk for breast cancer and perceptions of genetic testing in a sociodemographically diverse sample.

Informed consent for genetic testing for breast-ovarian cancer susceptibility requires that women understand basic concepts about the inheritance of cancer susceptibility and the benefits and risks associated with genetic testing. Women awaiting routine medical services (N = 220) were surveyed about their knowledge of breast cancer and cancer genetics and their perceptions of genetic testing and personal risk. There were no racial differences in median income or mean level of education. Compared to Caucasian women, African American women knew significantly less about breast cancer and about genetic risk for breast cancer. African American women had different psychological, social, and economic concerns as evidenced by how they weighted the benefits and risks of genetic testing. This study is the first to assess several dimensions of informed consent for genetic testing among a sociodemographically diverse group. The findings should enable health professionals to target the African American and lower-income populations with the appropriate education and counseling.

Smoking cessation programs for adolescents.

Many smoking cessation programs for adult smokers are described in the medical and nursing literature. Programs designed to prevent smoking among adolescents and children also are prevalent in the literature. Despite these prevention programs, many adolescents choose to start smoking. Once adolescents begin smoking, it is difficult to find ways to help them quit. Few programs exist that are targeted to help this population with smoking cessation. This article provides an in-depth review of 5 smoking cessation programs designed for adolescents. Each of the programs presents unique strategies for helping teenage smokers quit. Techniques used in these programs include peer leadership, nicotine patch therapy, peer support, computer instruction, and one-on-one counseling with a nurse practitioner. Each program was studied for efficacy with adolescents. Although none of the programs reviewed showed remarkable success, they serve as guides for future program development. Additional programs need to be developed and studied with larger, more diverse populations. Nurses must identify or develop smoking cessation programs that meet the needs of all types of adolescents and are effective in helping them to quit. Once designed, these smoking cessation programs should be made accessible to adolescents in a variety of settings.

Comparison of interleukin-1 beta expression by in situ hybridization in monoclonal gammopathy of undetermined significance and multiple myeloma.

We investigated whether interleukin-1beta (IL-1beta) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1beta appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1beta was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1beta mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1beta message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1beta mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1beta production. In the future, continued follow-up of IL-1beta positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1beta is predictive of those MGUS patients that will eventually progress to active myeloma.

The role of interleukin-1 beta in the pathogenesis of multiple myeloma.

Interleukin-1 beta has potent OAF activity, can increase the expression of adhesion molecules, and can induce paracrine IL-6 production (see Fig. 1). These biologic effects of IL-1 beta closely parallel several of the clinical features of human myeloma, such as osteolytic bone lesions, homing of myeloma cells to the bone marrow, and IL-6-induced cell growth. The increased production of adhesion molecules could explain why myeloma cells are found predominantly in the bone marrow. These fixed monoclonal plasma cells could subsequently stimulate osteoclasts through the production of IL-1 beta and paracrine generation of IL-6, resulting in osteolytic disease. Also, IL-6 produced by either a paracrine or autocrine mechanism can support the growth of the myeloma cells that may be manifested clinically by an elevated labeling index. In the future, continued follow-up of IL-1 beta-positive and IL-1 beta-negative MGUS patients should determine whether aberrant expression of IL-1 beta by monoclonal plasma cells is a critical genetic event in the progression of MGUS to myeloma. Because MGUS is relatively common in the general population and myeloma is incurable in almost all cases, identification of MGUS patients who are likely to progress to active myeloma will be important in the development of new therapeutic strategies. For example, an effective chemopreventive agent that prevents or delays the transition from MGUS to myeloma could have a major effect on the treatment of patients with monoclonal gammopathies.

Bone marrow and peripheral blood involvement in mantle cell lymphoma.

The peripheral blood smears, bone marrow aspirates and biopsies of 46 patients with mantle cell lymphoma were reviewed. The diagnosis of mantle cell lymphoma was established in all cases on extramedullary tissue samples using standard morphologic, phenotypic and molecular genetic criteria. 27/35 patients (77%) had circulating lymphoma cells (median 200%m of all circulating white blood cells; range 5-90%) identified by morphology at some point during the course of their disease. No statistical difference in survival was detected in patients with or without peripheral blood involvement. Lymphoma was identified in bone marrow aspirate specimens from 33/40 patients (83%) and in bone marrow biopsy specimens from 39/43 patients (91%). The pattern of marrow biopsy involvement was nodular (31 cases; 82%), interstitial (19 cases; 50%), paratrabecular (17 cases, 45%) and diffuse (12 cases; 32%). Although the median survival of patients with > or = 50% bone marrow involvement was 13 months, and the median survival of patients with < or = 50% was 49 months; no statistically significant differences between these small subgroups were observed. Mantle cell lymphoma frequently involves the peripheral blood and bone marrow. Its appearance is distinctive but variable, and immunophenotypic studies as well as morphologic confirmation by a biopsy of tissue other than bone marrow is still required for diagnosis.

Contrast in cytokine expression between patients with monoclonal gammopathy of undetermined significance or multiple myeloma.

We investigated whether differences in IL-6 and IL-1beta expression could be detected in monoclonal plasma cells from patients with MGUS or MM. Expression of IL-6 and IL-1beta in bone marrow cells was determined using cell sorting to enrich for plasma cells followed by reverse transcriptase/polymerase chain reaction (RT/PCR). Nineteen patients (six MGUS, two primary amyloid (AL), 11 MM) were studied. IL-6 mRNA expression was detectable in the sorted CD38+/CD45- plasma cell populations from 0/6 MGUS, 0/2 AL and 5/11 MM patients. All five MM patients with autocrine IL-6 expression demonstrated an elevated plasma cell labeling index. IL-1beta mRNA was detectable in the sorted CD38+/CD45- plasma cell populations from 1/6 MGUS, 0/2 AL and 10/11 MM patients. In situ hybridization (ISH) confirmed that the IL-1beta producing cells were plasma cells. In conclusion, autocrine production of IL-6 parallels a high labeling index and aberrant expression of IL-1beta correlates with the diagnosis of MM. Follow-up of IL-1beta-positive MGUS patients will determine whether aberrant expression of IL-1beta will predict those MGUS patients that will eventually progress to MM.

Sequence, expression and function of an mRNA encoding a soluble form of the human interleukin-6 receptor (sIL-6R).

Soluble receptors have been shown to be potent immunomodulators of their respective ligands. Since IL-6 is a central growth factor for myeloma cells, an sIL-6R may modulate IL-6 activity. We have previously reported a novel IL-6R mRNA from myeloma cells that exhibits a 94-nt deletion of the entire transmembrane domain from codons 356 (G-TG) to 387 (AG-G). The transmembrane domain deletion results in a shift in the translational reading frame with the insertion of 10 new amino acids followed by a stop codon. Sequence analysis shows the ligand-binding domain of the sIL-6R to be identical to that of the membrane-bound IL-6R up to the transmembrane domain deletion. The sIL-6R cDNA was expressed in QT-6 fibroblasts and PA-1 ovarian cells using the expression vector pCDM8. Supernates were immunoprecipitated with anti-IL-6R antibody and cells transfected with the sIL-6R cDNA produced a single band with a molecular weight of 50-55 kDa. This molecular weight corresponds to the size of the sIL-6R protein observed in normal human urine. Supernates were collected from mock or sIL-6R transfected PA-1 cells after 48 hours and assayed for their ability to stimulate or suppress the growth of an IL-6 dependent cell line, ANBL-6. Soluble IL-6R alone had no effect on the growth of the ANBL-6 cells. However, the growth of ANBL-6 cells by sIL-6R was potentiated in the presence of IL-6 and could be blocked by anti-IL-6 antibody. The above results suggest that, in the presence of IL-6, sIL-6R associates with gp130 leading to signal transduction and cell growth.

Detection of TSH receptor RNA in cultured fibroblasts from patients with Graves' ophthalmopathy and pretibial dermopathy.

Fibroblasts are target cells for the autoimmune process in Graves' ophthalmopathy and pretibial dermopathy. Because the autoantigen involved in the hyperthyroidism of Graves' disease is the TSH receptor, we sought to determine whether RNA encoding this receptor might be present in retroocular and pretibial fibroblasts. RNA was reverse transcribed and the resulting cDNA was amplified by the polymerase chain reaction using primers spanning a region of the extracellular domain of the human TSH receptor. The predicted amplified product, verified by direct sequencing, was detected when RNA was derived from fibroblasts, but not from the nonfibroblast cells studied. The demonstration in fibroblasts of RNA encoding this important autoantigen in Graves' disease suggests that the TSH receptor might play a role in the pathogenesis of the connective tissue manifestations of this disease.

Synthesis of renin by tubulocystic epithelium in autosomal-dominant polycystic kidney disease.

Evidence suggests an important role for the renin-angiotensin system in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). Therefore, we studied the presence of immunoreactive renin in renal biopsies and measured the concentrations of renin in cyst fluids. Normal kidneys and kidneys with renal artery stenosis were used for comparison. In ADPKD, immunoreactive renin was present in juxtaglomerular apparatus, associated arterioles, and in some cells within the connective tissue surrounding the cysts. Vascular immunoreactive renin was less prominent than in renal artery stenosis. Increased amounts of tubular immunoreactive renin were noted in polycystic kidneys, as compared to normal kidneys and kidneys with renal artery stenosis. Cyst fluids contained renin detected by Western analysis and enzymatic activity; concentrations were greater in gradient cysts than in nongradient cysts. Seventy-four percent of the renin in gradient cysts was active as compared to 23% in nongradient cysts and 15% in plasma. To determine whether cyst epithelial cells are capable of synthesizing renin, these cells were isolated in tissue culture. Enzymatic assay of extracts from these cells revealed the presence of renin-like enzymatic activity (1.3 +/- 0.8 ng AI/mg protein/hr). The synthesis of renin by tubulocystic epithelium was confirmed by [35S]-methionine radiolabeling of cyst-derived cells, followed by immunoprecipitation and SDS-PAGE and by detection of renin mRNA by the polymerase chain reaction. These results indicate that the tubulocystic epithelium has the potential to synthesize renin. Elevated levels of active renin in renal cysts may be linked to the pathogenesis of hypertension in ADPKD. The occurrence of renin in the lining epithelium of cyst walls raises the possibility that abnormal expression of the renin-angiotensin system may, by a paracrine or autocrine mechanism, regulate epithelial hyperplasia in growing renal cysts.

Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.

Molecular analysis and repair of a defective Ef alpha gene.

The molecular basis for the defective expression of Ef alpha was determined by analysis of the 5' region of a full length Ef alpha gene. The gene was isolated from a genomic library prepared from the A.CA/SnDv mouse strain. DNA sequence analysis of the 5' portion of the Ef alpha gene, which encodes the 5' regulatory sequences and the signal peptide, revealed the presence of a stop codon in the exon encoding the signal peptide. The remainder of the sequences were highly related to sequences found in previously characterized, functional E alpha alleles. Previous studies indicate that the f allele is transcribed at rates comparable to the rates of functional alleles and that mRNA accumulates in the cytoplasm. Primer extension analysis demonstrated that Ef alpha transcripts initiate identically to the functional Ek alpha allele, mapping the defect in the Ef alpha gene 3' of the transcriptional initiation site. To determine whether the stop codon in the signal peptide was the only major defect in this gene, reciprocal chimeric genes were constructed in which the 5' regions, including the first exons, of the defective f allele and the functional k allele were exchanged. The hybrid genes were inserted into an SV40 promotor driven expression vector for cotransfection with an Ek beta gene. Surface I-E expression was demonstrated using I-E specific mAb in Cos-7 and L cell lines transfected with the hybrid gene consisting of the 5' region of the k allele and the 3' portion of the f allele. Therefore, the single stop codon present in the exon encoding the leader peptide of the Ef alpha gene appears to be the only defect preventing this gene from expressing a functional E alpha-chain.

Transcriptional control of MHC class II antigen expression on mouse T cell lines.

We have analysed the factors which regulate MHC class II expression in mouse T cell lines. Two such lines, BW 5147 and PLT-24.2, were used in this study. Using 5-azacytidine (5 AzaC) we have shown that hypomethylation of DNA can induce class II antigen synthesis in BW 5147. The expression of class II in PLT-24.2 cells seems to be under a different control mechanism. Southern blot analysis of I-A beta gene in PLT-24.2 suggests that the expression of class II in this cell line is probably the outcome of a gene rearrangement. We hypothesise that insertion of viral long terminal repeats (LTR) next to the class II genes in transformed T cell lines can act as a promoter for the expression of class II antigens.

The flow and composition of pulmonary and systemic lymph in dogs with edema.

In animals with edema, pulmonary and systemic lymphatics may function to remove accumulated extravascular fluid in addition to responding to events as they occur in the exchanging vessels. We tested this hypothesis by measuring the flow rate and composition of thoracic duct and right duct lymph in anesthetized dogs made edematous by rapid fluid infusion. During a fluid challenge equivalent to 30% of body weight, thoracic and right duct lymph flow rates increased 30- and 60-fold, respectively. After the infusion, lymph flow rates rapidly decreased even though the dogs were edematous. During the postinfusion period, the decrease in right duct lymph flow rate was directly related to a decrease in estimated net pulmonary fluid filtration pressure. We conclude that lymph flow rate and composition reflect events occurring in the microvasculature whether or not edema is present.