A common nonsense mutation in EphB2 is associated with prostate cancer risk in African American men with a positive family history.

BACKGROUND: The EphB2 gene was recently implicated as a prostate cancer (PC) tumour suppressor gene, with somatic inactivating mutations occurring in approximately 10% of sporadic tumours. We evaluated the contribution of EphB2 to inherited PC susceptibility in African Americans (AA) by screening the gene for germline polymorphisms. METHODS: Direct sequencing of the coding region of EphB2 was performed on 72 probands from the African American Hereditary Prostate Cancer Study (AAHPC). A case-control association analysis was then carried out using the AAHPC probands and an additional 183 cases of sporadic PC compared with 329 healthy AA male controls. In addition, we performed an ancestry adjusted association study where we adjusted for individual ancestry among all subjects, in order to rule out a spurious association due to population stratification. RESULTS: Ten coding sequence variants were identified, including the K1019X (3055A-->T) nonsense mutation which was present in 15.3% of the AAHPC probands but only 1.7% of 231 European American (EA) control samples. We observed that the 3055A-->T mutation significantly increased risk for prostate cancer over twofold (Fisher's two sided test, p = 0.003). The T allele was significantly more common among AAHPC probands (15.3%) than among healthy AA male controls (5.2%) (odds ratio 3.31; 95% confidence interval 1.5 to 7.4; p = 0.008). The ancestry adjusted analyses confirmed the association. CONCLUSIONS: Our data show that the K1019X mutation in the EphB2 gene differs in frequency between AA and EA, is associated with increased risk for PC in AA men with a positive family history, and may be an important genetic risk factor for prostate cancer in AA.

Clinical characteristics of African-American men with hereditary prostate cancer: the AAHPC Study.

INTRODUCTION: The African-American Hereditary Prostate Cancer (AAHPC) Study was designed to recruit African-American families fulfilling very stringent criteria of four or more members diagnosed with prostate cancer at a combined age at diagnosis of 65 years or less. This report describes the clinical characteristics of a sample of affected AAHPC family members. METHODS: In all, 92 African-American families were recruited into the study between 1998 and 2002. Complete clinical data including age and PSA at diagnosis, number of affected per family, stage, grade, and primary treatment were available on 154 affected males. Nonparametric Wilcoxon two-sample tests and Fisher's exact test (two-tailed), were performed to compare families with 4-6 and >6 affected males with respect to clinical characteristics. RESULTS: The mean number of affected men per family was 5.5, with a mean age at diagnosis of 61.0 (+/-8.4) years. Age at diagnosis, PSA and Gleason score did not show significant differences between the two groups of families. Based on the Gleason score, 77.2% of affected males had favorable histology. Significantly, there were marked differences between the two groups in the frequency of node-positive disease (P=0.01) and distant metastases (P=0.0001). Radical prostatectomy was the preferred primary therapy for 66.2% of all affected men followed by 20.8% who chose radiation therapy. CONCLUSIONS: Our findings suggest that affected males who carry the highest load of genetic factors are at the highest risk for early dissemination of disease, thus efforts at early diagnosis and aggressive therapeutic approaches may be warranted in these families. Since the primary therapy choices in our study favored definitive treatment (87.0%) when compared to the 1983 and 1995 SEER data in which 28 and 64% received definitive treatment, respectively, it appears that affected African-American men in multiplex families may be demonstrating the reported psycho-social impact of family history on screening practices and treatment decisions for prostate cancer.

Prostate carcinoma knowledge, attitudes, and screening behavior among African-American men in Central Harlem, New York City.

BACKGROUND: Although the benefits of prostate carcinoma screening in reducing mortality rates have not been proven or shown to be cost-effective, screening, particularly using prostate specific antigen (PSA) tests, is widespread. A better understanding of screening behavior, knowledge of prostate carcinoma risk, and attitudes toward screening among men at high risk, such as African-American men, would be valuable. METHODS: A prevalence survey was conducted using 2 samples of African-American men, aged 50-74 years: a clinic sample drawn from all clinics in Central Harlem (n = 404) and a random-digit dial sample from the same geographic region (n = 319). The prevalence of self-reported PSA screening was estimated using a cognitive survey methodology based on the internal consistency of answers to four different questions. Prevalence estimates were adjusted to take into account the high proportion of nontelephone residences. RESULTS: The clinic sample, representing a poorer, more ill population (as determined by MOS Physical Function Scores, was less likely to report PSA screening than the community sample (11.1% in clinic sample vs. 25.5% in community). The prevalence of PSA testing in Central Harlem overall in this age group by using two different techniques was estimated to be 24%. In multiple logistic models, self-reported PSA screening was associated with age, education, favorable attitudes toward screening, and knowing someone who had prostate carcinoma. However, the association between these factors and the likelihood of self-reported PSA screening differed between clinic and community samples. CONCLUSIONS: The prevalence of self-reported PSA screening in Central Harlem was lower than that reported for other populations. These findings may be useful in the design of health education campaigns and for counseling innercity, low-income African-American patients appropriately about the disease.

African-American heredity prostate cancer study: a model for genetic research.

A genome-wide scan of high-risk prostate cancer families in North America has demonstrated linkage of a particular marker to Chromosome 1q (HPC1). An even greater proportion of African-American families have shown linkage to HPC1. Therefore, investigators at the National Human Genome Research Institute (NHGRI) in collaboration with Howard University and a predominantly African-American group of urologists established the African-American Hereditary Prostate Cancer (AAHPC) Study Network to confirm the suggested linkage of HPC in African Americans with a gene on Chromosome 1. Blood samples from recruited families were sent to Howard University for extraction of DNA. The DNA was sent to NHGRI at NIH where the genotyping and genetic sequence analysis was conducted. Genotype data are merged with pedigree information so that statistical analysis can be performed to establish potential linkage. From March 1, 1998, to June 1, 1999, a total of 40 African-American families have been recruited who met the study criteria. Preliminary results suggest that racial/ethnicity grouping may affect the incidence and extent of linkage of prostate cancer to specific loci. The importance of these findings lays in the future treatment of genetic-based diseases.

African-American heredity prostate cancer study: a model for genetic research.

A genome-wide scan of high-risk prostate cancer families in North America has demonstrated linkage of a particular marker to Chromosome Iq (HPC11. An even greater proportion of African-American families have shown linkage to HPC 1. Therefore, investigators at the National Human Genome Research Institute [NHGRI] in collaboration with Howard University and a predominantly African-American group of urologists established the African-American Hereditary Prostate Cancer (AAHPC) Study Network to confirm the suggested linkage of HPC in African Americans with a gene on Chromosome 1. Blood samples from recruited families were sent to Howard University for extraction of DNA. The DNA was sent to NHGRI at NIH where the genotyping and genetic sequence analysis was conducted. Genotype data are merged with pedigree information so that statistical analysis can be performed to establish potential linkage. From March 1, 1998, to June 1, 1999, a total of 40 African-American families have been recruited who met the study criteria. Preliminary results suggest that racial/ethnicity grouping may affect the incidence and extent of linkage of prostate cancer to specific loci. The importance of these findings lays in the future treatment of genetic-based diseases.

Recruitment experience in the first phase of the African American Hereditary Prostate Cancer (AAHPC) study.

The African American Hereditary Prostate Cancer (AAHPC) Study is an ongoing multicenter genetic linkage study organized by Howard University and the National Human Genome Research Institute (NHGRI), with support from the Office for Research on Minority Health and the National Cancer Institute. The goals of the study are to: (i) look for evidence of involvement of chromosome 1q24-25 (HPC1) in African American men with hereditary prostate cancer (HPC) and (ii) conduct a genome-wide search for other loci associated with HPC in African American men. To accomplish these goals, a network has been established including Howard University, the NHGRI, and six Collaborative Recruitment Centers (CRCs). The CRCs are responsible for the identification and enrollment of 100 African American families. To date, 43 families have been enrolled. Recruitment strategies have included mass media campaigns, physician referrals, community health-fairs/prostate cancer screenings, support groups, tumor registries, as well as visits to churches, barber shops, and universities. By far, the most productive recruitment mechanisms have been physician referrals and tumor registries, yielding a total of 35 (81%) families. Approximately 41% (n = 3400) of probands initially contacted by phone or mail expressed interest in participating; the families of 2% of these met the eligibility criteria, and 75% of those families have been enrolled in the study, indicating a 0.5% recruitment yield (ratio of participants to contacts). As the first large-scale genetic linkage study of African Americans, on a common disease, the challenges and successes of the recruitment process for the AAHPC Study should serve to inform future efforts to involve this population in similar studies.

The influence of gender on incidence and outcome of patients with bladder cancer in Harlem.

Although African Americans have a lower incidence of bladder cancer, overall survival is worse compared with American whites. This phenomenon has been attributed to the higher incidence of advanced disease at diagnosis and poor follow-up. Fifty-nine cases of bladder cancer were identified through the Tumor Registry at Harlem Hospital and reviewed retrospectively. Complete data were obtained for 42 patients. The primary independent variables of interest were primary care utilization, comorbid conditions, social variables, and gender. The outcome variables of interest were stage of disease at presentation and death. The median age at diagnosis in this group was 73 years compared with 68 for bladder cancer patients in the United States. There was no statistically significant correlation between primary care utilization or severity of comorbidities, and clinical stage at presentation. Similarly, these variables did not influence the occurrence of death as an outcome. For women, the mean age at diagnosis was 74.2 years compared with 67.3 in men (P = .112). The ratio of male-to-female cases in this group was 1.3 to 1 compared with 2.7 to 1 for the general US population. Women had lower odds of being diagnosed with superficial disease (OR = 0.24, 95% CI, 0.06-0.94) and a higher incidence of a cancer-specific death (OR = 2.7, 95% CI). The poor outcome and high incidence of bladder cancer cases among women in Harlem is intriguing. Overall, primary care utilization, comorbidities, and other social factors did not seem to influence stage or death as an outcome. The significantly elevated prevalence of smoking among women in this community, increased age at diagnosis, and possible environmental influences may play a role.

2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression.

2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA. These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H. Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin. Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture.

Acute urinary retention secondary to urethral inflammation from a vaginal contraceptive suppository in a 17-year-old boy.

We report the case of a 17-year-old boy who developed acute urinary retention following unprotected intercourse. His partner employed for the first time a nonoxynol-9-based commercial vaginal contraceptive insert. During intercourse the patient felt severe burning pain in the urethra. He was subsequently unable to void. Flexible cystourethroscopy revealed gross mucosal erythema and inflammation in the distal urethra and navicular fossa. We discuss the clinical management and review relevant literature.

PIP: Reported is the case of a 17-year-old US boy who developed acute urinary retention due to severe urethral inflammation, secondary to absorbance of a nonoxynol-9-based contraceptive. He had a recent history of unprotected intercourse with his regular sex partner until she used, for the first time, a vaginal suppository containing nonoxynol-9. During intercourse on this occasion, the adolescent experienced severe burning pain in the urethra and was subsequently unable to void. He denied any prior history of urinary tract infection, sexually transmitted diseases, or urethral discharge prior to this episode. The only significant clinical findings at examination were an inflamed meatal mucosa and severe tenderness to palpation 2 cm proximal to the glans. Flexible cystourethroscopy revealed gross mucosal erythema and inflammation in the distal urethra and navicular fossa. A French Foley catheter was easily inserted into the bladder and 1000 cm of clear urine were drained. An indwelling catheter was kept in place for 48 hours until the patient voided successfully. This is the first reported case of severe urethritis and obstruction in a young male. In this case, urethral absorption of nonoxynol-9 caused a severe inflammatory reaction sufficient to obstruct the distal urethra. When evaluating young men with acute urinary retention, clinicians should inquire about recent use of contraceptive inserts.

Antisense gene suppression against human ICAM-1, ELAM-1, and VCAM-1 in cultured human umbilical vein endothelial cells.

Antisense gene suppression has been carried out for human ICAM-1, ELAM-1, and VCAM-1 in cultured human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide, tumor necrosis factor alpha, or interleukin-1 beta. A panel of antisense phosphorothioate oligodeoxyribonucleotides (PS-ODN), complementary to mRNA or pre-mRNA of these molecules, were tested for their gene suppression activity monitored by radioimmunoassay of the respective cell surface adhesion molecules. Sequences targeted by effective antisense PS-ODNs were located throughout the mRNA and pre-mRNA. "Hot spots" of gene suppression sites for each region were observed. Shift of the PS-ODN hybridizing site upstream or downstream by a few bases resulted in drastic change of gene suppression efficiency. In addition to translation arrest and RNase H activity, a third mechanism was proposed for antisense gene suppression, involving multiple binding sites for PS-ODN and the activities of RNase H and RNases other than RNase H. Suppression of ICAM-1, ELAM-1, or VCAM-1 in HUVEC by their antisense PS-ODNs resulted in the reduction of adhesion of monocytes and U937 to HUVEC. This may suggest cooperativity among the adhesion molecule pairs in endothelial-leukocyte adhesion, since decrease of a single adhesion molecule on EC surface significantly reduced cell-cell adherence.

(2'-5')-Oligo-3'-deoxynucleotides: selective binding to single-stranded RNA but not DNA.

Oligodeoxynucleotides with (2'-5') internucleotide linkages have been synthesized on a solid support via standard cyanoethyl phosphoramidite chemistry. This simple change in the oligonucleotide bond connectivity led to unique properties. UV melting temperature experiments indicate that the (2'-5')-oligo-3'-deoxyadenylates, (2'-5')-3'-dA8 and (2'-5')-3'-dA8(s) phosphorothioate, hybridize selectively to single-stranded RNA but not DNA. The complex (2'-5')-3'-dA8:poly (U) (Tm = 32 degrees C) was nearly as stable as the natural (3'-5')-2'-dA8 and poly (U) (Tm = 33 degrees C) in 130 mM NaCl, and 10 mM phosphate buffer (pH 7.5). However, no association was observed upon mixing (2'-5')-3'-dA8 and poly (dT). The (2'-5') linkages also confer greater resistance to exo- and endonucleolytic degradation compared with (3'-5')-linked oligomers. The rate of degradation of (2'-5')-3'-dA8 was almost four times less than that of (3'-5')-2'-dA8 in cell culture medium containing 10% heat-inactivated fetal calf serum. An increase in stability for (2'-5')-3'-dA8 against endonuclease activity was observed in both cytoplasmic and nuclear extracts. The nucleic acid selectivity of (2'-5')-oligo-3'-deoxynucleotides may represent an important design feature to improve the efficacy of antisense oligonucleotides.

The presence of peptidoglycan-polysaccharide complexes in the bowel wall and the cellular responses to these complexes in Crohn's disease.

Interestingly, using a monoclonal antibody, peptidoglycan-polysaccharide complexes (PPC) were detected intracellularly in the mucosa and submucosa of the bowel wall of Crohn's disease (CD) patients. PPC are the main constituents of the gram-positive bacterial cell wall. These PPC were however detected in the normal bowel wall also. Therefore, in this study the hypothesis that an enhanced immune responsiveness to bacterial antigens plays a pivotal role in the induction or the chronicity of CD was tested. As antigens, the peptidoglycan structures of intestinal bacteria (Eubacterium aerofaciens or fecal PPC) or of Streptococcus pyogenes, the 65-kDa heat shock protein and muramyl dipeptide (MDP), the smallest bioactive subunit of peptidoglycan, were used. The proliferative responses of peripheral blood (PB) mononuclear cells (MNC) of healthy subjects and patients in a remissive stage of CD or an active CD stage were examined. Of this last patient group the MNC responses of the mesenterial lymph nodes that drain the inflamed gut area were measured also. The responses of PB-MNC of the healthy subjects and the patients in a remissive CD stage were not different. Compared to the responses in remissive CD, the PB-MNC responses in active CD to the eubacterial cell wall and streptococcal cell wall antigen were significantly higher. At the inflammation site in active CD, the lymph nodes, the responses to most of the bacterial antigens were significantly higher than in the PB. In summary, the results show the presence of bacterial peptidoglycan in the bowel wall and the immune responsiveness, especially at the inflammation site, to these antigens in active CD and therefore present suggestive evidence for the role of peptidoglycan in the etiology and/or pathogenesis of CD.

Glycoprotein of human immunodeficiency virus type 1 synthesized in chronically infected Molt3 cells acquires heterogeneous oligosaccharide structures.

Diversity of oligosaccharide structures on the glycoprotein of HIV-1 was studied in individual clones of Molt3 cells chronically infected with HIV-1IIIB. A glycoprotein of molecular weight 140 kD (gp140) was found to be shed into the medium from one of these clones, which unlike normally processed gp120, contained significant proportions of endo H resistant oligosaccharides. Treatment of infected cells with the inhibitors of oligosaccharide trimming enzymes affected the glycosylation pattern as well as the secretion of the glycoprotein into the medium. The exposure of the principal neutralizing domain (PND) on the surface of gp140, as measured by its accessibility to thrombin cleavage, was comparable to that observed with gp120. Sera obtained from mice inoculated with purified gp140 contained high titered anti-V3 antibodies and blocked HIV-1IIIB-induced syncytium formation. These results demonstrate that although glycosylation of viral glycoproteins is governed by the host cell glycosyl transferases, glycoprotein secreted from biological clones of the same host cells acquires different oligosaccharide structures. Exposure and immunogenicity of the PND in one such glycosylation variant are comparable to the normally processed gp120 molecule.

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.

In vitro toxicological evaluation of ISIS 1082, a phosphorothioate oligonucleotide inhibitor of herpes simplex virus.

ISIS 1082, a phosphorothioate oligonucleotide targeted to a translation initiation codon of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) virion capsid protein UL13 inhibits in vitro viral replication. To better understand the pharmacological properties of ISIS 1082, we examined its effects in nonvirally infected HeLa cells by using a number of cytotoxicity assays. Our data indicate that ISIS 1082 had no effect on HeLa cell viability as measured by cellular proliferation and clonogenic assays at concentrations as high as 100 microM. Additionally, DNA, RNA, and protein synthesis were only inhibited by 25% in cells treated with 100 microM ISIS 1082. The effects of ISIS 1082 on DNA synthesis were compared with those of acyclovir and trifluorothymidine, two clinically used antiherpetic agents. Acyclovir displayed effects similar to that of ISIS 1082. However, trifluorothymidine, which has been reported to be a potential mutagen and teratogen, significantly altered DNA replication at concentrations from 1 to 100 microM. Isolated HeLa DNA polymerases were inhibited by the compound, with a 50% inhibitory concentration of 2 microM. The in vitro antiviral (K. Draper and V. Brown-Driver, submitted for publication; K.G. Draper and V. Brown-Driver, Antiviral Res. Suppl. 1:106, 1991) and cytotoxicity studies suggest that ISIS 1082 is a selective, nontoxic, antiherpetic therapeutic agent.

Characterization of a neutralizing monoclonal antibody to the external glycoprotein of HIV-1.

The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal antibody and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gp120 from HIV-1IIIB, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia normally observed when uninfected cells are cocultured with HIV-1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-1-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gp120 known to contain a neutralizing epitope, this epitope was localized between amino acids 307 and 320 (V3 loop) in the external glycoprotein molecule. The monoclonal antibody did not interfere with the binding of gp120 to CD4, or with the subsequent step of CD4-induced shedding of gp120 from the viral envelope. However, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.

Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.

Brefeldin A inhibits the processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1.

The processing and secretion of the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected T cells and in primary macrophages treated with an antiviral antibiotic brefeldin A (BFA). BFA blocks the egress of proteins from the endoplasmic reticulum and has a profound effect on the structure of cis/medial Golgi. In MOLT-3 cells infected with the IIIB strain of HIV-1 (MOLT-3/IIIB), BFA inhibited the intracellular processing of gp160. The secretion of envelope proteins from these cells was significantly inhibited in the presence of BFA. The gag proteins, on the other hand, were processed and secreted normally. BFA also inhibited the proteolytic processing of gp160 in primary macrophages infected with HIV-1. The infectivity of virus pelleted from the medium of MOLT-3/IIIB cells treated with BFA was markedly lower than that obtained from untreated cells. These results demonstrate that the proteolytic processing of gp160 in HIV-1-infected cells takes place after the glycoprotein exists the endoplasmic reticulum and that the transport of glycoprotein to the cell surface is required for assembly of complete HIV-1 particles.

Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro.

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid-phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells.