Posttransplant lymphoproliferative disorders in lung transplant patients: the Cleveland Clinic experience.

PTLD is a well-recognized complication of organ transplantation. Large series of heart, renal, and liver transplants have been examined for the incidence and behavior of PTLD. However, reports of the incidence and characteristics of PTLDs in lung transplant (LTx) patients are few. We report our experience with PTLDs in a large series of LTx recipients at a single institution and compare them to other solid organ transplant recipient PTLDs seen at our institution. Twenty-eight patients were found to have PTLD, of whom 8 were lung transplant recipients. We evaluated nine PTLD specimens from these 8 patients for their histology, immunophenotype (CD20, CD3, EBV-LMP1), EBER status by in situ hybridization, and clinical features. The incidence of PTLD was 3.3% (8/244 patients). The time to development of PTLD, after transplant, was short (median time, 7 mo). All were of B-cell lineage. Overall, EBV was demonstrated in 77.7% (7 of 9 specimens) of PTLDs. All specimens tested for clonality were found to be monoclonal. Five patients died, with a median time to death of only 4.6 months. PTLDs in LTx patients are EBV-associated B-cell, predominantly monoclonal lymphoid lesions similar to other solid organ transplant PTLDs. Compared with other solid organ transplant recipients with PTLD at our institution, PTLDs in LTx patients have a propensity to involve the transplanted organ (P =.001, Fisher's exact test), occur earlier after transplant (P =.003, Wilcoxon test), and have a shorter survival (P =.002, log rank test). Reasons for this may include the relatively higher level of immunosuppression required in these patients and limited options in decreasing it. Although the incidence is low, careful early monitoring of lung transplantation patients is warranted because of the poor prognosis of patients developing this complication.

Typical and atypical chronic lymphocytic leukemia differ clinically and immunophenotypically.

We compared the features of 17 cases of atypical chronic lymphocytic leukemia (aCLL) with those of a clinical control group of 24 cases of CLL. Quantitative flow cytometric data, available for 12 cases, were compared with an immunophenotypic control group of 58 cases using a relative fluorescence indexfor CD5, CD23, CD79b, and surface immunoglobulin light chain (sIg). Compared with the clinical control group, patients with aCLL had a higher mean WBC count and a lower platelet count. Patients with aCLL had a significantly higher probability of disease progression. Compared with an immunophenotypic control group of 58 CLL cases, 12 cases of aCLL demonstrated significantly higher expression of CD23. There was no significant difference in expression of sIg, CD79b, or CD5 between the groups. CD38 expression was noted in only 1 (9%) of 11 tested cases; 2 (18%) of 11 cases had trisomy 12. aCLL can be distinguished from typical CLL morphologically, clinically, and immunophenotypically. Atypical morphologic features in CLL seem to be a marker of aggressive clinical behavior.

Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: apparent immunohistochemical false-positives do not get the message.

BACKGROUND: Several studies have reported what seem to be false-positive results using the Food and Drug Administration (FDA)-approved HercepTest (Dako Corp, Carpinteria, CA) to profile Her-2/neu amplification and overproduction in breast carcinoma. False-positive status has been based on comparisons with gene copy enumeration by fluorescence in situ hybridization (FISH) and with comparisons to immunohistochemistry (IMH) results using a monoclonal antibody. However, simple overexpression by tumor cells that have normal gene copy has not been evaluated by profiling mRNA expression, ie, such cases could simply represent true-positive, transcriptionally upregulated overexpression. MATERIALS AND METHODS: Four hundred infiltrating ductal carcinomas of breast were evaluated by IMH using monoclonal (CB11; Ventana Medical Systems, Inc, Tucson, AZ) and polyclonal (HercepTest; Dako) antibodies after antigen retrieval (AR). A polyclonal antibody sans AR (PCA/SAR) was also used. All IMH stains were evaluated and scored according to the guidelines for the FDA-approved HercepTest. A total of 145 of 400 carcinomas were subsequently evaluated by direct and digoxigenin-labeled (Dig) FISH, and 144 of 400 were evaluated by detection of mRNA overexpression via autoradiographic RNA:RNA in situ hybridization. RESULTS: Overall HercepTest/CB11 IMH discordance was 12%. Expression of mRNA was highly concordant with FISH and DigFISH amplification and with CB11 and PCA/SAR immunohistology. IMH false-positive cases (no Her-2/neu gene amplification) occurred with both HercepTest (23%) and CB11 (17%), and the majority of false-positive results (34 of 44) were scored as 2+. All 2+ false-positive cases were mRNA-negative. Combined results of HercepTest and CB11 showed that 79% (38 of 48) of 3+ cases were Her-2/neu gene amplified, but only 17% (seven of 41) of 2+ cases had increased gene copy. CONCLUSION: Discordant HercepTest/FISH results, and to a lesser extent discordance with CB11 IMH, are most commonly false-positive results with a score of 2+. The 2+ score as defined in the guidelines for the FDA-approved HercepTest should not be used as a criterion for trastuzumab therapy unless confirmed by FISH. Determination of Her-2 gene copy number by FISH may be a more accurate and reliable method for selecting patients eligible for trastuzumab therapy.

Use of novel t(11;14) and t(14;18) dual-fusion fluorescence in situ hybridization probes in the differential diagnosis of lymphomas of small lymphocytes.

Increasingly, molecular biologic techniques have become important in the diagnosis of non-Hodgkin lymphomas. In the differential diagnosis of lymphoma(s) of small lymphocytes (LSL), reliable detection of t(11;14) or t(14;18) would confirm the diagnosis of mantle cell lymphoma (MCL) or follicle center lymphoma (FCL), respectively. A total of 87 LSL cases (27 MCL, 39 FCL, 17 small lymphocytic lymphoma [SLL], 3 marginal zone lymphomas, and 1 paraimmunoblastic variant of SLL) were diagnosed by a combination of light microscopy, immunohistochemistry, and flow cytometric immunophenotyping. Interphase fluorescence in situ hybridization (FISH) for t(11;14) and t( 14;18) using dual-fusion probes (Vysis, Downers Grove, IL) was performed on touch (n = 69) or gravity (n = 18) preparations from these cases. Of 27 MCL cases tested, 25 (93%) had demonstrable t(11;14), none had t(14;18), and 2 were negative for t(11;14) and t(14;18). Twenty-five of 39 (64%) FCL cases had t(14;18), none had t(11;14), and the remaining FCL cases (14 cases [35%]) had neither t(11;14) nor t(14;18). All 17 (100%) SLL cases had neither t(11;14) nor t(14;18). All 3 (100%) marginal zone lymphoma cases had neither t(11;14) nor t(14;18). The case of paraimmunoblastic variant of SLL had t(11;14) and was negative for t(14;18). No discrepant [i.e., positive for both t(11;14) and t(14;18)] or false-positive cases were noted. Interphase FISH using these commercially available probes is a useful adjunct to light microscopy, immunohistochemistry, and flow cytometric immunophenotyping in the diagnosis of LSL. FISH can be performed successfully on archival single-cell preparations (touch preparations or gravity preparations) when fresh tissue is unavailable. No discordant or false-positive cases were identified.

Validation of a multicolor interphase fluorescence in situ hybridization assay for detection of transitional cell carcinoma on fresh and archival thin-layer, liquid-based cytology slides.

OBJECTIVE: To evaluate the feasibility of performing multicolor interphase fluorescence in situ hybridization (FISH) on ThinPrep slides of transitional cell carcinoma (TCC). STUDY DESIGN: Slides from 20 voided urine specimens were prepared by the ThinPrep technique (Cytyc, Boxborough, Massachusetts, U.S.A.), pretreated using a pretreatment kit and subjected to hybridization with the multicolor FISH probe UroVysion (Vysis, Downers Grove, Illinois, U.S.A.). Archival slides were placed in xylene, destained in alcohol and washed prior to pretreatment. Urines from patients with cytology-positive, biopsy-proven grade 1 (n = 5), 2 (n = 7) and 3 (n = 5) TCC and negative cytology and biopsy (n = 3) were selected. Freshly prepared (n = 10) and archival (n = 10) slides were used. RESULTS: All carcinoma cases were FISH positive (> 5 cells with complex abnormalities of > or = 2 studied chromosomes per slide). None of the normal samples were aneusomic. Gain of chromosomes 3, 7 and 17 constituted the majority of positive cases. Proper destaining and slight decrease in stringency wash conditions enabled reliable detection of signals in archival cases. CONCLUSION: Routine ThinPrep slides can be used for multicolor interphase FISH analysis of urine cytology specimens. Archival slides provide the opportunity to analyze by FISH the nature of atypical cells identified by cytology. This revised method allows FISH technology more accessibility for routine use in cytology laboratories.

Aneusomy of chromosomes 7, 8, and 17 and amplification of HER-2/neu and epidermal growth factor receptor in Gleason score 7 prostate carcinoma: a differential fluorescent in situ hybridization study of Gleason pattern 3 and 4 using tissue microarray.

Recent evidence shows that the proportion of poorly differentiated prostate carcinoma (Gleason pattern [GP] 4/5) is a surrogate factor for biochemical failure after radical prostatectomy (RP). However, little is known about specific molecular and cytogenetic changes in this aggressive component of localized prostate cancer. We constructed a tissue microarray containing areas of GP 3 and 4 from formalin-fixed radical prostatectomy specimens of 39 patients with Gleason score 7 carcinoma (>or=50% GP 4), known pathologic staging parameters (stage < T3b), and biochemical failure data (mean follow-up, 30 months; range, 5 to 74 months). Interphase fluorescent in situ hybridization (FISH) was performed on 5-microm microarray sections using pericentromeric probes to chromosomes 7, 8, and 17 and probes for the HER-2/neu and epidermal growth factor receptor (EGFR) genes. Low-level amplification of HER-2/neu was found in 26% of cases (3 to 5 signals per nucleus, corrected for chromosome 17 aneusomy). Aneusomy of chromosomes 7, 8, and 17 was identified in 21%, 15%, and 5% of cases, respectively. All aberrations occurred almost exclusively in GP 4 carcinoma (8 of 8 aneusomies 7, 2 of 2 trisomies 17, 9 of 10 HER-2/neu amplifications, and 5 of 6 aneusomies 8; P < .001). The presence of HER-2/neu amplification was associated with high tumor volume (>2.0 cm(3), P = 0.004). Among patients with negative surgical margins, gain of chromosome 7 was associated with biochemical failure after RP (P =.004, log-rank). Amplification of the EGFR gene occurred in only 1 case (3%). Significant differences in HER-2/neu amplification and gain of chromosomes 7, 8, and 17 were detected between GP 4 prostate carcinoma and GP 3. The frequency of aberrations increased with tumor volume. Chromosome 7 abnormalities may play an important role in cancer progression in margin-negative patients. EGFR amplification was rare, suggesting that this oncogene is not altered at the gene copy number level.

Clinicopathologic reassessment of primary cutaneous B-cell lymphomas with immunophenotypic and molecular genetic characterization.

Primary cutaneous B-cell lymphomas (PCBLs) may have particular clinicopathologic characteristics distinct from their lymph node-based counterparts. It has been suggested that PCBLs should have a separate classification system. The aim of this study was to determine whether the Revised European-American Lymphoid Neoplasms (REAL) classification is applicable to PCBL. Thirty-nine cases of PCBL from 36 patients, consisting of 20 men and 16 women (median age 66 yrs), were included in this study. Paraffin-section immunohistochemistry for CD3, CD5, CD10, CD20, CD43, Bcl-2, Bcl-6, and cyclin D1 was performed in all cases. Immunostaining for immunoglobulin light chains was also performed on cases histologically diagnosed as extranodal marginal zone lymphoma (MZL) and primary cutaneous B-cell lymphoma unclassifiable (PCBLu). Polymerase chain reaction (PCR) analysis of t(14;18) was performed in all cases. Immunoglobulin heavy chain gene rearrangement (VDJ) was tested by PCR on all follicle center lymphoma (FCL), MZL, and PCBLu cases. The 39 cases consisted of 15 (39%) FCLs, 13 (33%) diffuse large B-cell lymphomas (DLCL), 9 (23%) extranodal MZL, and 2 cases of PCBLu. Anatomically, 59% of PCBLs occurred in the head and neck, of which approximately 57% were FCL. Five of six cases presenting on the lower extremity were DLCL. Follow-up data was available from all 39 patients with a mean of 50.8 months. All but two patients are alive with or without disease at last contact. One patient with DLCL died of lung metastases and the other DLCL patient died of sepsis as a complication of therapy. In all 15 cases of FCL, CD10 and/or Bcl-6 expression supported the follicle center origin of the neoplastic cells. In contrast to previous reports, we found that 53% (8 of 15) of primary cutaneous FCL had either Bcl-2 protein expression or t(14;18). Our data indicate that many cases of primary cutaneous FCL have Bcl-2 alterations similar to their nodal counterpart. We found that 95% (37 of 39) of PCBLs could be classified according to the REAL classification, supporting its applicability in cutaneous lymphomas.

Concomitant oncoprotein detection with fluorescence in situ hybridization (CODFISH): a fluorescence-based assay enabling simultaneous visualization of gene amplification and encoded protein expression.

We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

The effect of fixation on detection of B-cell clonality by polymerase chain reaction.

It has been suggested that neutral buffered formalin (NBF)-fixed, paraffin-embedded, or fresh specimens might provide satisfactory DNA templates for polymerase chain reaction (PCR) assays used in establishing the clonality and presumptive B-cell lineage of lymphoma. The suitability of other fixatives used by hematopathologists, such as B5, is still undetermined. Thirty cases were identified from the files of the Cleveland Clinic Foundation, Cleveland Ohio, that showed abnormal immunoglobulin heavy chain (IgH) rearrangement by Southern blot analysis (SBA). Corresponding paraffin-embedded tissue samples fixed in NBF (21 cases), B5 (18 cases), Hollande's fixative (17 cases), zinc formalin (ZF) (5 cases), and Bouin's fixative (3 cases) were studied. With use of consensus primers against the framework 3 (FR3) and FR2 regions of the VH gene, paired against JH primer(s), PCR analysis was performed. bcl-2/IgH translocation was also studied. Ten reactive lymphoid samples were used as controls, and 40 cases were evaluated. Successful amplification of a clonal proliferation was manifested as one or two discrete narrow bands in the appropriate size range. The sensitivity of detecting clonality was 95, 94, 67, 80, and 0% for NBF, Hollande's fixative, B5, ZF, and Bouin's fixative, respectively. Although NBF and Hollande's fixative were 100% specific, consistent false-positive results were a major problem with B5-fixed tissue. Paraffin-embedded tissue, fixed in NBF, Hollande's fixative, and ZF solutions, may be used for DNA extraction and PCR assays for establishing B-cell clonality. The precipitating fixative B5 and Bouin's solution should not be used for this purpose until the issue of false-positive results is resolved.

Comparative analysis of interphase FISH and RT-PCR to detect bcr-abl translocation in chronic myelogenous leukemia and related disorders.

The t(9;22)(q34;q1 1) between the abl and bcr genes plays a pivotal role in the diagnosis and pathogenesis of chronic myelogenous leukemia (CML). Its detection is routinely accomplished by Southern blot analysis and karyotyping. Interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) are emerging molecular techniques that offer viable alternatives. We analyzed 40 samples of peripheral blood and bone marrow (CML, 16; acute myelogenous leukemia, 6; acute lymphoblastic leukemia [ALL], 1; chronic lymphoblastic leukemia, 2; myelodysplasias, 4; myeloproliferative disorders, unclassified, 3; nonleukemic hematologic malignancies, 3; hypercellular bone marrow, 1; normal control samples, 2; and K562 cell line samples, 2) for the presence of bcr-abl fusion gene and its messenger RNA (mRNA) transcript by FISH and RT-PCR, respectively. We compared the results with results of Southern blot analysis and karyotyping when available. Cost analysis was performed. Thirty-three samples were evaluable by FISH; 14 of 14 evaluable CML samples and one ALL sample were positive for bcr-abl by FISH (100%). The other 15 evaluable samples were negative; 16 of 16 (100%) and 13 of 16 (81%) of CML cases were positive for bcr-abl mRNA by RT-PCR (chemiluminescent blot method) and RT-PCR (colorimetric method), respectively. The ALL sample was positive by both RT-PCR methods. All other samples were negative by RT-PCR (chemiluminescent blot method), and all but 1 case of myeloproliferative disorder tested negative by RT-PCR (colorimetric method). We conclude the utility of FISH and RT-PCR is associated with certain limitations, such as insufficient RNA for RT-PCR and the occasional absence of internal positive FISH control signals. However, each procedure offers (with a high concordance rate) a specific and cost-effective alternative to Southern blot analysis and karyotyping and improved turnaround time for the detection of bcr-abl fusion gene or its mRNA transcript.

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

The use of amplified variable number of tandem repeats (VNTR) in the detection of chimerism following bone marrow transplantation. A comparison with restriction fragment length polymorphism (RFLP) by Southern blotting.

Chimerism analysis after allogeneic bone marrow transplantation (alloBMT) allows detection of early marrow engraftment, disease relapse, and graft rejection. Our objective was to do retrospective and prospective studies of chimerism analysis by restriction fragment length polymorphism (RFLP) by Southern blotting and variable number of tandem repeats (VNTR) by polymerase chain reaction (PCR) to compare and contrast the methods. The retrospective group comprised 46 samples from 26 patients previously analyzed by RFLP, while the prospective group contained 34 samples from 25 patients. Using four different VNTR primers (D1S80, D17S30, D1S111, and APO-B), the recipient and donor samples amplified by the PCR were screened for unique banding patterns. The VNTR primer with the unique banding pattern was used to detect chimerism in each sample. A total of 635 VNTR analyses were performed. Interpretation was blinded for previous RFLP results. A comparison between the VNTR and RFLP results and a cost analysis of the two procedures were done. A unique VNTR banding pattern was present in 49 of 51 patients (identical twins in one case). The VNTR analysis showed complete chimerism in 68 samples, mixed chimerism in 9, and recurrences in 2. This agreed with the RFLP results in 64 (80%) of 80 samples. Failure to detect 1% to 10% of recipient DNA accounted for 15 (VNTR, 8; RFLP, 7) discordances. Follow-up revealed all donor DNA in five cases, decreasing quantities of recipient DNA in two cases (six samples), and no additional studies available in four cases. In one case, VNTR detected a complete chimerism when the DNA was insufficient for RFLP analysis. The cost analysis revealed an approximately 50% savings with the use of VNTR; VNTR is a viable alternative to RFLP in the detection of chimerism after bone marrow transplantation and offers substantial cost savings, faster turnaround time, easier preparation of the DNA, smaller DNA requirements, and the elimination of radioisotopes and cumbersome restriction enzymes.

Activation of alveolar macrophage TNF and MCP-1 expression in vivo by a synthetic peptide of C-reactive protein.

Administration of multilamellar vesicles (MLV) encapsulating a synthetic peptide (RS-83277) derived from human C-reactive protein (CRP) augments anti-tumor activity of murine alveolar macrophages and reduces established pulmonary metastases of experimental tumors. To explore mechanisms involved in these phenomena, we investigated cytokine and integrin (CDllb) expression of bronchoalveolar lavage (BAL)-derived alveolar macrophages in control (blank MLV) and RS-83277-MLV-treated C57BI mice. Alveolar macrophage production of tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant bioactivity increased at 48 h after treatment with RS-83277-MLV but not control MLV. Chemoattractant activity was neutralized by antibody to monocyte chemoattractant protein-1 (MCP-1), but not irrelevant immunoglobulin G(IgG). Changes were reflected by augmented TNF-alpha and MCP-1 mRNA levels in pulmonary tissue and enhanced CD11b expression on mononuclear leukocytes derived from total lung tissue, but not on BAL-derived alveolar macrophages. Results suggest that RS-83277-MLV treatment is associated with activation of alveolar macrophage TNF-alpha and MCP-1 production and up-regulation of adhesion molecules on pulmonary mononuclear leukocytes but not on alveolar macrophages.

Human astrocyte growth regulation: interleukin-4 sensitivity and receptor expression.

We previously reported that interleukin-4 (IL-4) inhibited proliferation of a human astrocytic cell line derived from non-neoplastic adult cortex. To determine whether this effect was receptor-associated and/or limited to only non-neoplastic astrocytes, we examined IL-4 responsiveness and receptor expression in human astrocytic cell lines derived from three different sources: non-neoplastic cerebral cortex (lines P1N, P2N, W3N); neoplastic low grade astrocytoma (LGA) (lines FRLGA, RTLGA); and highly malignant glioblastoma multiforme (GBM) (lines STTG1, CRTG2, WITG3, RUTG4). All lines except RUTG4 GBM expressed IL-4 receptor mRNA. Proliferation and DNA synthesis were markedly suppressed by IL-4 in a dose- and time-dependent manner in all non-neoplastic astrocyte and LGA lines, but not (0/4) GBM. This negative growth-regulatory effect of IL-4 was blocked by specific antibody to human IL-4 receptor but not by irrelevant IgG. In contrast, IL-4 stimulated interleukin-6 (IL-6) secretion in non-neoplastic astrocytes and LGA as well as in GBM cells expressing IL-4 receptor; secretion was undetectable in RUTG4 GBM which did not express receptor. These results indicate that: (i) responsiveness to IL-4 occurs in both non-neoplastic and neoplastic human astroglia; (ii) responsiveness is associated with IL-4 receptor expression; and (iii) sensitivity to negative growth signalling by IL-4 occurs selectively in astrocytes from non-neoplastic cortex or low grade neoplasia but not from highly malignant GBM.

Human monocytes produce monocyte chemoattractant protein 1 (MCP-1) in response to a synthetic peptide derived from C-reactive protein.

We reported previously that a synthetic peptide (RS-83277) derived from human C-reactive protein (CRP) augmented human monocyte/macrophage tumoricidal activity and cytokine production. RS-83287, a synthetic peptide derived from a different CRP site, was ineffective. Because chemoattractant properties have been attributed to some CRP-derived peptides, we hypothesized that RS-83277, in addition to activating effects, might promote human monocyte chemotaxis. Results indicated that neither CRP peptide RS-83277 nor RS-83287 was, itself, a chemoattractant. RS-83277, but not RS-83287, however, elicited time-dependent production of monocyte chemoattractant activity in conditioned media (CM) of cultured human mononuclear leukocytes and purified, adherent monocytes (MO). CM from nonadherent MO contained no activity, indicating that adherence was required for monocyte response. Monocyte chemoattractant activity was dose-dependent and was removed by treatment with immobilized antibody to human monocyte chemoattractant protein 1 (MCP-1) but not by irrelevant IgG. These results indicate that a specific peptide segment of CRP acts upon human adherent monocytes to promote production of the autocrine chemotactic and activating factor MCP-1. Data suggest that degraded CRP represents a complex source of biologically active peptides which, among other effects, may amplify monocyte recruitment to sites of injury.

Regulation of monocyte chemoattractant protein-1 expression in adult human non-neoplastic astrocytes is sensitive to tumor necrosis factor (TNF) or antibody to the 55-kDa TNF receptor.

Infiltration of the central nervous system (CNS) by monocytes is a characteristic of many non-malignant disease processes, although the signals regulating such traffic are unclear. Tumor necrosis factor (TNF) and other inflammatory cytokines have been shown to elicit production of monocyte chemoattractant activity in glioma cells, but the regulation of such activity in non-neoplastic adult astrocytes has not been examined. We previously observed that TNF constituted a proliferative signal for non-neoplastic adult human astrocytes in vitro involving the 55-kDa TNF receptor. In the present study, we demonstrate that TNF exposure enhances the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA and functional monocyte chemoattractant activity in non-neoplastic astrocytes. Results indicated that MCP-1 mRNA expression was maximal within 3 h, and was further augmented by the protein synthesis inhibitor cycloheximide (CY). Antibody (htr-9) directed against the 55-kDa TNF receptor also elicited MCP-1 mRNA expression while antibody to the 75-kDa TNF receptor (utr-1) was ineffective. Secretion of monocyte chemoattractant activity was significantly greater in TNF- or htr-9-treated astrocytes than in utr-1-treated or untreated controls; activity was abolished by treatment with antibody to MCP-1. These findings suggest that non-neoplastic adult human astrocytes may contribute to CNS inflammatory responses by mediating recruitment of peripheral blood monocytes.

Use of resealed erythrocytes as delivery system for C-reactive protein (CRP) to generate macrophage-mediated tumoricidal activity.

We have shown previously in several mouse tumor systems that multilamellar vesicles (MLV) are an effective delivery system for generation of macrophage-mediated tumoricidal activity by C-reactive protein (CRP). Here we show that resealed erythrocyte ghosts (red cell ghosts, RCG) can function in the same manner. CRP associated with red cell ghosts (CRP-RCG) inhibited established lung metastases of T241 fibrosarcoma in C57B1/6J mice. The degree of inhibition was comparable to that observed with CRP-MLV. In other experiments, peritoneal exudate cells, obtained from mice pretreated with CRP-RCG i.p., inhibited growth and pulmonary metastases of T241 tumor in the Winn neutralization assay. Similar results were obtained with MCA-38 colon carcinoma in the Winn assay. These studies indicate that erythrocytes deserve consideration as another delivery system for biological response modifiers.