Neutrophil priming by cigarette smoke condensate and a tobacco anti-idiotypic antibody.

A polyphenol-rich reagent, referred to as CSC, was isolated from cigarette smoke condensate and shown to prime purified human neutrophils. A mouse monoclonal anti-idiotypic antibody directed against the polyphenol-reactive determinants on a rabbit polyclonal anti-tobacco glycoprotein antibody was generated and shown to also prime neutrophils. After priming by CSC or tobacco anti-idiotypic antibody, there was a 2.5-fold to threefold increase in CD11b/18 expression and doubling of the number of formyl-methionyl-leucyl-phenylalanine receptors on the cells. The primed cells showed a twofold increase, compared with unprimed cells, in production of superoxide and release of neutrophil elastase after stimulation with formyl-methionyl-leucyl-phenylalanine. Neutrophils in peripheral blood of cigarette smokers have been shown to be primed and more responsive to activating agents. The priming effects attributed to whole cigarette smoke have been demonstrated in these studies using purified neutrophils and CSC or tobacco anti-idiotypic antibody. These studies are a first step in testing the hypothesis that the inflammatory process contributing to progression of chronic obstructive pulmonary disease in ex-smokers may be driven, in part, by tobacco anti-idiotypic antibodies. This hypothesis is novel and carries with it the implication of a heretofore unrecognized autoimmune component in the disease process manifested through production of anti-idiotypic antibodies with tobacco-like activity.

A new PhD training track: a proposal to improve basic science teaching.

There has been increasing criticism of medical basic science teaching; much of this has focused on overcrowding of the curriculum, inadequate application to clinical medicine, and the limited commitment of the faculty to teach. We have analyzed some of the factors that may contribute to these complaints, such as the fragmentation of physiology and the conflicting roles of the medical basic scientist. We have also reviewed some previous suggestions for improving basic science teaching. We suggest that a basic scientist with a background of integrative physiology, pharmacology, anatomy, and pathology, with a special emphasis on pathophysiology, would be well qualified to assume an important role in the medical education of the future. Because there is at present no established training program of this type, we have proposed a PhD training track with this objective and have listed some of the advantages and disadvantages of such a program.

Activation of the classical pathway of complement by tobacco glycoprotein (TGP).

Tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from tobacco leaves, activates the classical complement pathway through a mechanism that appears to involve direct interaction with C1q. A binding site on C1q for TGP can be localized by competitive inhibition with DNA to a region located in the junction between the collagen-like and globular regions of the molecule. A protein with activity similar to TGP has also been isolated from cigarette smoke condensate (TGP-S); it shares a binding site on C1q with TGP and has similar functional activity, with the exception that complement activation does not proceed to formation of a C3 cleaving enzyme. The ability of TGP and TGP-S to activate complement can be partially duplicated using polyphenols associated with tobacco leaf and smoke, i.e., chlorogenic acid and rutin. These polyphenols also compete with TGP for a binding site on immobilized C1q, suggesting that the polyphenol portion of TGP is critical for activation of complement. These results provide an additional mechanism for complement activation by cigarette products that, in vivo, could result in a localized complement depletion, generation of biologically active complement cleavage products, and initiation of an inflammatory response.

Flow cytometric measurement of proliferation-associated nuclear antigen P105 and DNA content in immuno-proliferative small intestinal disease (IPSID).

Immunoproliferative small intestinal disease (IPSID), most common in Mediterranean countries, is characterized by lymphomatous infiltration of the small intestine and is usually associated with the synthesis of anomalous immunoglobulin alpha heavy chains. Flow cytometric analysis of DNA content, S phase fraction, and quantitative analysis of the proliferation-associated nuclear antigen, P105, were performed in 23 patients with IPSID to determine if they could be used as prognostic indicators in this disease. Eighteen patients had low-grade, two had intermediate-grade, and three had high-grade lymphoma. Eight patients had clinical stage IE disease, 12 had stage IIE, and three had stage IIIE disease. Eleven patients survived > 5 yr (good prognosis), four survived between 2-5 yr (intermediate prognosis), and eight survived 2 yr or less (poor prognosis). The S phase fraction of patients with poor prognosis was significantly higher than those with intermediate or good prognosis (P < 0.004). Flow cytometric evaluation of S phase fraction may offer important prognostic information in patients with IPSID and could be useful in the clinical management of patients with this highly variable clinical syndrome. Further studies evaluating the value of DNA flow cytometry in larger groups of patients with IPSID are warranted.

Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose.

Eight patients who had hematologic relapse of chronic myelogenous leukemia (CML) after undergoing allogeneic bone marrow transplantation (BMT) were treated with leukocyte infusions from the original bone marrow donors. All patients had previously received marrow grafts from HLA-identical siblings. Six patients were in the accelerated phase of their disease and two were in blast crisis. Each patient received a predetermined T-cell dose within a narrow range of 2.5 to 5.0 x 10(8) T cells/kg. Three patients also received short courses of therapy with alpha interferon to control elevated white blood cell counts within the first several weeks after leukocyte transfusions. Seven of eight evaluable patients developed graft-versus-host disease (GVHD) at a median of 32 days after the initial infusion. One patient had fatal GVHD. A second patient had grade 3 acute GVHD, which has responded to immunosuppressive therapy. The remaining patients all had mild grade I GVHD. Six patients continue to require modest doses of prednisone more than 6 months after infusion. Four patients developed marrow aplasia, which in three patients required marrow boosts from the original donors. Two of these three patients have normal hematopoietic function, whereas the third patient remains growth factor and transfusion dependent. Both patients treated in blast crisis have died, one from GVHD and one from disease progression. All six patients in the accelerated phase are alive and in cytogenetic remission at a median of 42 weeks after infusion. Five of these six patients are in molecular remission. This study demonstrates that leukocyte infusions that administered a defined T-cell dose can exert a profound graft-versus-leukemia effect and are an effective form of salvage immunotherapy in allogeneic marrow transplant recipients. This therapeutic approach appears to be a viable alternative to existing chemotherapeutic and immunomodulatory strategies for the treatment of relapsed CML.

DNA flow cytometry of sebaceous cell carcinomas of the ocular adnexa: introduction to the technique in the evaluation of periocular tumors.

DNA content abnormalities are well recognized in tumor cell biology and kinetics. We introduce the technique of DNA flow cytometry through the study of the sebaceous cell carcinomas of the ocular adnexa. By correlating the data to the standard histopathologic parameters of pagetoid changes, degree of anaplasia, and stromal inflammation, significant associations are revealed. All aneuploid tumors demonstrate pagetoid spread, and more severe anaplasia and stromal inflammation. All diploid tumors are nonpagetoid and have lesser degrees of anaplasia and stromal inflammation. A complete review of the technique with a discussion of the implications and applicability to the study of ocular adnexal tumors is presented.

Failure of T cell receptor-anti-CD3 monoclonal antibody interaction in T cells from marrow recipients to induce increases in intracellular ionized calcium.

There are multiple immune defects in T cells from recipients after bone marrow transplantation (BMT). This study examines recipient T cells for increases in intracellular ionized calcium concentration [( Ca2+]i) after binding the T cell receptor-CD3 complex with anti-CD3 MAb. PBL from 10 of 23 short-term recipients (less than 1 yr after BMT) responded poorly (less than 35% of control) to anti-CD3 stimulation and PBL from 9 of 23 had blunted calcium flux responses (35-70% of control). Purified CD2+, CD56- cells from seven additional short-term recipients including three autologous marrow recipients were closely examined, and a sizable proportion of CD3+ cells from six of seven recipients did not increase [Ca2+]i after anti-CD3 stimulation. The decreased magnitude of the responses was due to decreased numbers of responding cells and not to a decrease in mean CD3 fluorescent intensity or in calcium flux responses on a single cell basis. Five of seven long-term recipients (greater than 1 yr after BMT) had PBL that responded normally and two of seven had PBL with blunted calcium flux responses. The data show that the signal transduction response mediated by the CD3-antigen receptor as measured by calcium flux is defective early after autologus or allogeneic BMT.

Image analysis confirmation of DNA aneuploidy in flow cytometric DNA distributions having a wide coefficient of variation of the G0/G1 peak.

With nuclei extracted from paraffin-embedded tissues, resolution of normal diploid DNA and abnormal near-diploid/aneuploid populations by flow cytometry (FCM) is especially difficult. These samples, compared with fresh tissue, tend to show a broader DNA distribution, appearing as a wide (high) coefficient of variation (CV) of the G0/G1 peak. To address the question of whether there may be aneuploid populations hidden in wide CV diploid G0/G1 peaks, the authors measured DNA content in nuclei extracted from paraffin-embedded tumor tissue by morphometric image analysis (IA) in addition to FCM. Of 29 samples showing little evidence of DNA aneuploidy by FCM, in 20 of 20 with G0/G1 CVs greater than or equal to 5.50% there was an aneuploid population when analysis was performed by IA. Of the remaining nine samples with CVs less than or equal to 4.41%, all were diploid in the G0/G1 region by both FCM and IA. The presence of aneuploid populations in FCM distributions with wide CV G0/G1 peaks can be confirmed by IA.

Increased density of HLA-DR antigen on monocytes of patients infected with the human immunodeficiency virus.

Monocytes and macrophages play an important role in the pathogenesis of the acquired immunodeficiency syndrome (AIDS). Dysfunction of these cells contributes to the immunocompromised state that characterizes AIDS, and they probably serve as a reservoir for the human immunodeficiency virus (HIV). HIV-infected macrophages may also be responsible for the infection of many CD4-positive lymphocytes by means of cell to cell contact during the initiation of immunological responses. The efficiency of this process would be enhanced by activation of the macrophages, since that is accompanied by increased expression of class II major histocompatibility (HLA-DR) antigen. Using a direct blood antibody marking procedure in conjunction with flow cytometry, we have analyzed the expression of HLA-DR antigen on the surfaces of monocytes present in the peripheral blood of HIV-infected patients. In contrast to the results reported by other investigators who purified the monocytes using Ficoll-Hypaque centrifugation prior to antibody marking, we found that the percentage of monocytes expressing HLA-DR antigen was identical in the patients and normal controls. However, a subpopulation of monocytes was detected in the blood of the majority of the patients that was expressing increased levels of HLA-DR antigen. It was also found that the proportion of monocytes with a high density of HLA-DR antigen on their surfaces negatively correlated with the absolute numbers of CD4-positive lymphocytes present in the peripheral blood of the patient. These findings support the postulated role of monocytes and macrophages in the HIV infection and ultimate destruction of CD4-positive lymphocytes.

Monoclonal antibodies to acetylcholine receptor secreted by human x human hybridomas derived from lymphocytes of a patient with myasthenia gravis.

Peripheral blood lymphocytes of a patient with myasthenia gravis (MG) were fused to the non-secreting human lymphoblastoid line HuNSI to produce human x human hybridomas that secrete monoclonal antibodies (mAb) to acetylcholine receptor (AChR). Screening of hybridomas for antibody production involved an enzyme-linked immunosorbent (ELISA) assay with AChR from Torpedo californica (TAChR). 25 of 302 wells tested (8.3%) were positive for anti-AChR antibody production and have been stable in their secretion of mAb for eleven months. Nine lines have been studied in detail. All produced IgM mAb, and most had greater activity against membrane-bound TAChR, than against solubilized TAChR. For anti-AChR clones, the mAb concentration in culture supernatants ranged from 2 to 33 micrograms/ml. Saturation curves of binding to TAChR performed on 4 lines demonstrated dissociation constants (Kds) estimated to range from 0.1-1.0 nM. The patient whose lymphocytes were used in this study had a serum anti-AChR antibody concentration of 243nM against human AChR and 15nM against AChR from T. californica. The results demonstrate the feasibility of producing stable human x human hybridomas secreting mAb to the autoantigen from the peripheral blood of patients with organ-specific autoimmune diseases. The mAb produced here may prove to be useful in analyzing, and possibly treating, the autoimmune phenomena in MG.

Relationships between alternative complement pathway activation, C-reactive protein, and pneumococcal infection.

In the absence of specific antibody, opsonization of Streptococcus pneumoniae may be mediated by the alternative complement pathway (AP) or by C-reactive protein (CRP) via C1 binding. To determine the role of these mechanisms in pneumococcal (PNC) disease, we studied 19 patients with differing severities of PNC infection. C4 and CRP levels and zymosan-induced consumption of 50% hemolytic complement (CH50) were measured in specimens obtained acutely and then weekly. In patients with complicated illness, the modified mean CH50 in acute sera was 178 +/- 57 U/ml, significantly lower than the mean CH50 of 331 +/- 80 U/ml in patients with uncomplicated illness (P less than 0.05). The values of the two groups on a given day approximated each other on days 7, 14, and 23. Consumption of complement by zymosan was also lower in acute sera of patients with complicated illness, with a mean value of 19 +/- 18 U/ml compared with 58 +/- 30 U/ml in those with uncomplicated illness (P less than 0.05). This difference was also seen on day 7 (P less than 0.05). Disease involving lower-numbered PNC serotypes (less than 10) correlated with reduced availability of AP factors in acute sera, independent of illness severity. Mean CRP levels were inversely related to zymosan-induced complement activation in patients with complicated illness. These data suggest that in vivo depletion of AP factors is significantly greater in patients with complicated illness and is associated with high CRP levels. CRP may enhance AP activation via C3 convertase generation and function with it as a preantibody host defense mechanism.

Complement activation in myasthenia gravis plasma by Torpedo acetylcholine receptor liposomes.

The purpose of this study was to determine if antibodies from patients with myasthenia gravis (MG) react in a biologically relevant manner with membrane bound Torpedo californica acetylcholine receptor (AChR). Antibody-mediated complement activation was measured in plasma from 15 patients with MG and 9 controls following incubation with Torpedo AChR-rich membrane vesicles (AChR liposomes). Significantly more (p less than 0.001) complement activation occurred in the patient samples compared with the controls. Torpedo AChR liposomes are well characterized and have been widely used to study the structure and function of AChR. These results suggest that Torpedo AChR liposomes may also be used in immunologic studies with autoantibodies from patients with MG.

Plasmapheresis with immunosuppressive drug therapy in progressive multiple sclerosis. A pilot study.

In light of encouraging preliminary data, 45 patients with severely progressive multiple sclerosis underwent long-term plasmapheresis in conjunction with low-dose cyclophosphamide and prednisone therapy. The disease progression was monitored by the Kurtzke disability status scale (DSS) and functional systems scale, neuro-ophthalmologic evaluations, evoked potentials, computed tomographic scans, and suppressor cell function assays. The conditions of 28 of the 45 patients improved significantly, the conditions of 14 patients showed limited improvement, and the conditions of three patients neither improved nor worsened. Improvement in other parameters correlated with the clinical results. Significant improvement in suppressor cell function was noted in those patients whose conditions had improved by one or more steps on the DSS.

Immune reactivity against membranes containing ganglioside GM1 in chronic-progressive multiple sclerosis: observation by spin-membrane immunoassay.

A spin-membrane immunoassay has been employed to examine the immune reactivity of whole serum from patients with chronic-progressive multiple sclerosis (CPMS) against liposomes containing ganglioside GM1. Exposure to serum resulted in complement-mediated lysis of the GM1-liposomes. No lysis occurred with liposomes devoid of ganglioside. The mean (+/- S.E.M.) lysis values were 52.6 (+/- 9.8)% for fifteen CPMS patients and 32.9 (+/- 7.2)% for nine controls. The difference between the means was highly significant (student's t-test, P less than 0.0001), indicating increased anti-ganglioside immunity in patients with CPMS.

Cellular response to human acetylcholine receptor in patients with myasthenia gravis.

A preparation of human skeletal muscle acetylcholine receptor (AchR) was used in vitro as an antigen to stimulate lymphocytes from patients with myasthenia gravis (MG). Clinical data obtained from the patients included duration and severity of disease; history of steroid treatment or prior thymectomy; and the presence of thymoma. Lymphocytes from patients with MG showed a significantly higher response to human AchR antigen than did lymphocytes from control subjects. Previous studies of cellular response to AchR have used receptor prepared from eel or ray electric organs. By stimulating lymphocytes from MG patients with a preparation of human AchR, we have come one step closer to documenting a possible contribution of a cellular immune response to the pathogenesis of MG.

Serologic studies in a family with heterozygous C2 deficiency.

Twelve family members of a patient with systemic lupus erythematosus (SLE) and heterozygous deficiency of the second component of complement (C2) were studied. Histocompatibility (HLA) typing was determined for A, B, and DR and MB antigens. Serum samples were tested for a variety of antinuclear antibodies (ANA), lymphocytotoxic antibodies and rheumatoid factors, and C2 levels were determined by hemolytic titration. Inheritance of C2D, the gene coding for C2, was limited to the haplotype HLA-A25, B18, DR2. Low but significant titers of ANA, rheumatoid arthritis nuclear antigen (RANA) and/or rheumatoid factors were found in eight of the nine adult family members without association with HLA haplotype. The sister of the proband had persistently strongly positive LE cell preparations for more than a decade and had joint pains while taking sulfa drugs. The son of the proband had leukemia. All other family members were healthy. We conclude that the increased incidence of rheumatic disease in persons with C2D deficiency is multifactorial and requires environmental factors or other hereditary factors unrelated to the HLA-A25, B18, DR2 haplotype. The C2D gene is clearly not associated with positive ANA tests or immunoprecipitins to RANA.