A preliminary study of a video intervention to inform solid organ transplant recipients about skin cancer.

PURPOSE: To obtain preliminary evidence on the effect of a skin cancer prevention video for adult solid organ transplant recipients (SOTR) and informational brochures on outcomes of skin cancer knowledge, beliefs, prevention and detection behaviors, and personal agency (self-confidence/personal control) for behaviors. BACKGROUND: SOTR have a high risk of skin cancer potentiated by life-long immunosuppressive therapy posttransplantation. Skin cancer in SOTR is aggressive and difficult to treat. Prevention and early detection are important for reducing risk and improving skin cancer outcomes, but methods to inform SOTR about their risk are understudied. METHODS: A brief, evidence-based skin cancer informational video tailored to SOTR was evaluated using a quasi-experimental design that compared the outcome variables in two groups of SOTR seen in 4 transplantation clinics within 4-6 weeks posttransplantation. The video/brochure group (VBG) viewed the video once and received skin cancer information brochures. The brochure group (BG) received brochures only. Participants completed a survey on sun protection behavior (6 items; alpha = 0.75), personal agency (6 items; alpha = 0.64), beliefs (6 items; alpha = 0.60), skin cancer knowledge (6 items), and skin self-examination (SSE; 1 item) at baseline and 3 months postintervention. Data were analyzed using descriptive statistics and 2 × 2 analysis of variance. RESULTS: Of 113 participants, 90 completed both surveys (VBG, n = 46; BG, n = 44). Both groups had a significant increase in sun protective behavior (P < .001), skin cancer knowledge (P < .001), beliefs (P = .003), and personal agency (P = .003). There was no effect of either intervention on SSE. CONCLUSION: Both interventions effectively informed SOTR about skin cancer and sun protection, promoted favorable beliefs, and improved personal agency, but were not differentially effective, suggesting that the addition of the video may not be necessary or that the video may need to be viewed more than once. More in-depth SSE teaching strategies may be necessary.

Maternal germ-line transmission of mutant mtDNAs from embryonic stem cell-derived chimeric mice.

We report a method for introducing mtDNA mutations into the mouse female germ line by means of embryonic stem (ES) cell cybrids. Mitochondria were recovered from the brain of a NZB mouse by fusion of synaptosomes to a mtDNA-deficient (rho degrees ) cell line. These cybrids were enucleated and the cytoplasts were electrofused to rhodamine-6G (R-6G)-treated female ES cells. The resulting ES cell cybrids permitted transmission of the NZB mtDNAs through the mouse maternal lineage for three generations. Similarly, mtDNAs from a partially respiratory-deficient chloramphenicol-resistant (CAP(R)) cell line also were introduced into female chimeric mice and were transmitted to the progeny. CAP(R) chimeric mice developed a variety of ocular abnormalities, including congenital cataracts, decreased retinal function, and hamaratomas of the optic nerve. The germ-line transmission of the CAP(R) mutation resulted in animals with growth retardation, myopathy, dilated cardiomyopathy, and perinatal or in utero lethality. Skeletal and heart muscle mitochondria of the CAP(R) mice were enlarged and atypical with inclusions. This mouse ES cell-cybrid approach now provides the means to generate a wide variety of mouse models of mitochondrial disease.

The roles of CD11/CD18 and ICAM-1 in acute Pseudomonas aeruginosa-induced pneumonia in mice.

Neutrophil accumulation in response to Pseudomonas aeruginosa in the lungs is mediated through CD11/CD18. This study determined the roles of CD11a, CD11b, and intercellular adhesion molecule (ICAM)-1 in P. aeruginosa-induced pneumonia and compared the function of ICAM-1 using Abs or ICAM-1 mutant mice. Anesthetized BALB/c mice pretreated with either Abs against CD11a, CD11b, ICAM-1, or rat IgG received intratracheal instillation of P. aeruginosa for 4 h. In other studies, ICAM-1 mutant and wild-type mice received either anti-ICAM-1 Ab or rat IgG followed by instillation of P. aeruginosa. The data show that Abs against CD11a, CD11b, and ICAM-1 in BALB/c mice inhibited neutrophil emigration by 79, 81, and 56%, respectively. ICAM-1 mutant mice showed no inhibition of neutrophil emigration compared with wild-type mice. Pretreatment with anti-ICAM-1 Ab inhibited neutrophil emigration in wild-type (129/SvxC57) mice by 67% but had no effect in ICAM-1 mutant mice, suggesting that the Ab was acting specifically through recognition of its Ag. We conclude that CD11a and CD11b are required for neutrophil emigration. The observed function of ICAM-1 varies depending on the method by which it is inhibited. Abs may overestimate function by altering other cellular functions or mutant mice may develop alternative pathways of emigration.

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Lymphocyte accumulation during Pseudomonas aeruginosa-induced pneumonia in rodents does not require CD11a and intercellular adhesion molecule-1.

During Pseudomonas aeruginosa-induced pneumonia in rodents, the acute infiltrate of neutrophils is followed by accumulation of lymphocytes in the perivascular connective tissue. The roles of the adhesion molecules CD11a/CD18 and intercellular adhesion molecule-1 (ICAM-1) in this accumulation of lymphocytes were investigated. The numbers of lymphocytes in P. aeruginosa-induced pneumonia were compared in animals treated with blocking antibodies to either CD11a, ICAM-1, IgG, or no antibody. In other experiments, the lymphocyte accumulation during P. aeruginosa-induced pneumonia in ICAM-1 mutant mice was compared with that in wild-type mice. In rats, both a murine anti-rat CD11a antibody and nonspecific murine IgG partially inhibited the lymphocyte accumulation by 30 to 40% compared with animals that received no antibodies. In mice, blocking antibodies to either CD11a or ICAM-1 did not decrease the lymphocyte accumulation compared with mice given IgG or no antibody. Further, there was no attenuation of the lymphocyte accumulation induced by P. aeruginosa in the ICAM-1 mutant mice compared with wild-type mice, either in the total number of lymphocytes or the number of CD4+, CD8+, or B cells. We conclude that neither CD11a/CD18 nor ICAM-1 are required for lymphocyte accumulation during P. aeruginosa-induced pneumonia in rodents. The partial inhibition of the lymphocyte accumulation in both the anti-CD11a- and IgG-treated rats may be due to nonspecific effects of foreign proteins on cellular functions.

Inflammatory and immune responses are impaired in mice deficient in intercellular adhesion molecule 1.

Gene targeting was used to produce mice deficient in intercellular adhesion molecule 1 (ICAM-1) or CD54, an immunoglobulin-like cell adhesion molecule that binds beta 2 integrins. Homozygous deficient animals develop normally, are fertile, and have a moderate granulocytosis. The nature of the mutation, RNA analysis, and immunostaining are consistent with complete loss of surface expression of ICAM-1. Deficient mice exhibit prominent abnormalities of inflammatory responses including impaired neutrophil emigration in response to chemical peritonitis and decreased contact hypersensitivity to 2,4-dinitrofluorobenzene. Mutant cells provided negligible stimulation in the mixed lymphocyte reaction, although they proliferated normally as responder cells. These mutant animals will be extremely valuable for examining the role of ICAM-1 and its counterreceptors in inflammatory disease processes and atherosclerosis.

Characterization of the murine Icam-1 gene.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a cell adhesion molecule that interacts with the leukocyte beta 2 integrins, LFA-1 and Mac-1. Murine inflammatory models are being utilized increasingly to define the role that ICAM-1 induction plays in the initiation of inflammation. We have isolated murine genomic clones that contain the Icam-1 gene including over 2 kb of 5' flanking sequence. The gene for murine Icam-1 spans over 13 kb and is composed of seven exons and six introns. Each of the extracellular immunoglobulin-like domains of ICAM-1 is encoded by a single exon that ends with the first base of the next codon. Examination of ICAM-1 expression in vivo shows that mRNA levels of ICAM-1 are low in all organs except for the lung but increase markedly in multiple organs at 3 h after administration of endotoxin. The 5' flanking region of the murine gene contains a putative TATA box and potential SP-1, AP-1, and AP-3 sites in positions nearly identical to those in the human gene.

An initiation codon mutation in CD18 in association with the moderate phenotype of leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by mutations in the CD18 gene which codes for the beta 2 integrin subunit. We studied two patients, the first of which had a moderate LAD phenotype and expressed only 9% of CD11/CD18 on blood leukocytes. RNA from lymphoblasts was reverse-transcribed, and the cDNA was amplified, cloned, and sequenced. An ATG to AAG alteration in the initiation codon was detected in 39 of 45 (87%) cDNA clones. This mutation was detected in the father, but not in the mother. The maternal defect was shown to be a frameshift mutation with the deletion of a single T in the aspartic acid codon at position 690 (GAT), 11 amino acids N-terminal to the beginning of the transmembrane domain. This mutation predicts a polypeptide which would terminate without transmembrane or cytoplasmic domains. The frameshift mutation was also found in the second patient who had the severe phenotype of LAD (less than 1% of CD11/CD18), indicating that this allele does not encode a functional protein. The partial expression in the patient with a moderate phenotype must be derived from the initiation codon mutation and may be due to a low level of initiation of translation of the CD18 mRNA at the second codon (CUG).

Characterization of eosinophils in a continuous ambulatory peritoneal dialysis patient with eosinophilic peritonitis.

A continuous ambulatory peritoneal dialysis patient developed eosinophilic peritonitis and was followed for 7 months. After 1 month, the peritonitis resolved, with a concomitant drop in percentage of hypodense eosinophils (Eos) recovered from peritoneal dialysate (PD) as well as a drop in fluid major basic protein levels. Blood eosinophil differential percentages were low, but the percentage of hypodense Eos in the blood tended to be relatively increased. Stool samples showed no evidence of parasitic infection, and epicutaneous skin tests were negative. Leukotriene C4 levels remained relatively constant as did white blood cell counts. Flow cytometric analysis of lymphocytes and granulocytes from PD and blood revealed high levels of CD23-positive lymphocytes.

Study of the effects of liposomal amphotericin B on Candida albicans, Cryptococcus neoformans, and erythrocytes by using small unilamellar vesicles prepared from saturated phospholipids.

We compared the anticellular effects of liposomal amphotericin B (AmB) formed from AmB and small unilamellar vesicles. The small unilamellar vesicles with or without cholesterol were prepared from three L-alpha-phosphatidylcholines with saturated acyl chains of different lengths: distearoyl (C18), dipalmitoyl (C16), and dimyristoyl (C14). We found that the anticellular potency of liposomal AmB, compared with that of free AmB, decreased with decreasing length of the acyl chain of the phospholipid and increased with the addition of cholesterol. In a parallel study (S. Jullien, A. Vertut-Croquin, J. Brajtburg, and J. Bolard, Anal. Biochem. 172:197-202, 1988), we found that binding of AmB to vesicles decreased with increasing length of the acyl chain of the phospholipid and decreased with the addition of cholesterol. We conclude that the anticellular effects of liposomal AmB preparations are due to the levels of AmB remaining free (unbound to the lipids) in these preparations.

Flow cytometric indirect immunofluorescence assay with high sensitivity and specificity for detection of antibodies to human immunodeficiency virus (HIV).

A flow cytometric immunofluorescence assay (FIFA) was developed to detect antibodies to human immunodeficiency virus (HIV) using live cell indirect immunofluorescence and analysis by flow cytometry. A panel of 107 sera, previously tested for anti-HIV antibody with the Abbott Enzyme-Linked Immunosorbent Assay test and Western blot (WB), was rescreened by FIFA. Antibodies were tested on HIV-infected and uninfected H9 cells in the FIFA. Although ELISA results indicated seven false positive results by comparison with the WB, 46 of 46 FIFA positive results tested WB positive and 61 of 61 FIFA negative results were WB negative. The results of FIFA showed 100% sensitivity and 100% specificity compared with WB. This rapid, quantitative, relatively easy assay makes FIFA an alternate confirmatory test for the presence of HIV antibodies.

Monoclonal antibodies to type D retrovirus (SRV-2).

Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.

Role of cell defense against oxidative damage in the resistance of Candida albicans to the killing effect of amphotericin B.

A laboratory-derived mutant of Candida albicans B311 (L) and a clinical isolate (C) of C. albicans, both lacking membrane ergosterol, were less susceptible to amphotericin B (AmB)-induced cell membrane permeability to K+ and lethality than was the wild-type laboratory strain (B311) which contained ergosterol. The resistance of L and C to AmB-induced killing was much greater than the level of resistance to AmB-induced cell membrane permeability. L and C were also less susceptible to killing by H2O2 than was B311, and when treated with menadione, they each produced less H2O2 than did B311. In addition, their levels of catalase activity were 3.8-fold (L) and 2-fold (C) higher than that of B311. The ergosterol deficiency in L and C probably impaired AmB binding to the cells, thereby lowering AmB effectiveness as measured by both cell membrane permeability and killing. Resistance of strains L and C to oxidation-dependent damage likely contributed to a diminished response to AmB-induced lethality.

Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule.

Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.

Cellular tropism of the human retrovirus HTLV-III/LAV. I. Role of T cell activation and expression of the T4 antigen.

In cultures of normal human lymphocytes infected with the human retrovirus HTLV-III/LAV, detectable cytoplasmic virus appears and then disappears in a proportion (1 to 10%) of cells, followed by release of virus detected by particulate reverse transcriptase activity, virus antigen assay, and infectivity titer. Virus infection is associated with loss of detectable T4 antigen on infected cells and, ultimately, complete loss of T4+ cells from the culture. Residual non-T4+ cells are not susceptible to a second infection with HTLV-III/LAV, and in cultures of separated cell populations, substantial virus replication occurred in T4+ T cells and minimally, if at all, in non-T4+ cells. We could not detect a disproportionate loss of cell surface phenotype (other than T4) in comparison of infected and noninfected cultures of lymphocytes or purified T4+ T cells when these cultures were monitored with a panel of monoclonal antibodies that detect the major mononuclear cell types (alpha-T11, alpha-T3, alpha-Mo2, alpha-B1), functional T cell subsets (alpha-T8, alpha-Leu-8, alpha-T17), or activated/proliferating cells (alpha-T10, alpha-Ia, alpha-T9, alpha-4F2, alpha-Tac). HTLV-III/LAV replication was quantitatively greatest in lymphocytes stimulated with phytohemagglutinin (PHA) and cultured in the presence of interleukin 2 (IL 2). Once activated by PHA, virus production in nondividing (irradiated) cells was similar to that in nonirradiated cells, but was substantially reduced if radiation was performed before PHA stimulation. Omission of PHA, IL 2, or both resulted in progressively lower amounts of virus replication. However, virus replication was detected and T4+ T cell depletion occurred in all cultures, regardless of medium supplement or radiation. T4+ T cells absorb infectious virus, and the binding of HTLV-III/LAV to the surface of T4+ T cells, but not to non-T4+ cells, was directly demonstrated. Binding is equivalent in activated and nonactivated cells and at 4 degrees and 37 degrees C. Reciprocal inhibition of binding was observed with alpha-T4a monoclonal antibody and virus. Exposure of cells to alpha-T4a before and during HTLV-III/LAV inoculation inhibited subsequent virus replication. We conclude that T4+ T cells are the major target for HTLV-III/LAV replication, that this tropism is related to expression of the T4 antigen that serves as a binding site for virus, that infection is inexorable in T4+ T cells regardless of subset or activation state, and that the activation/proliferative state of the cells is not a necessary determinant of infectivity, but rather, determines the amount of replication that will ensue.