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Three of 16 human monoclonal antibodies (hu-mAbs) enhanced human immunodeficiency virus type 1 (HIV-1) infection of MT-2 target cells by means of a mechanism that is dependent on complement. Enhanced infections are characterized by an increase in cytopathic effects and antigen synthesis as well as an increase in the production of progeny virus as detected by release of reverse transcriptase activity and infectious virus into the culture medium. Analyses by radioimmunoprecipitation, Western blot, and ELISA using the pENV9 envelope fragment localize the antigenic specificities of these three hu-mAbs to the N-terminal two-thirds of the transmembrane protein gp41. Competitive binding experiments indicate that the hu-mAbs are reactive with immunodominant epitopes of gp41 recognized by sera from essentially all HIV-1-infected subjects. Combination dose-effect experiments demonstrate that these hu-mAbs can act synergistically in vitro to enhance HIV-1 infection. These data demonstrate that hu-mAbs directed against the HIV-1 transmembrane glycoprotein gp41 can enhance HIV-1 infection in vitro. The availability of these reagents allows for the mapping of enhancing epitopes on HIV-1 and provides a means for studying whether deletion of such enhancing epitopes from candidate HIV-1 vaccines might improve the protective immune response to HIV-1 in immunized humans and chimpanzees.

Dextran sulphate (MW 5000 and 8000) and a polysulphated glycosaminoglycan (MW 10,000), at concentrations that provided complete protection in a homologous infection assay, failed to block syncytium formation and the resulting cytopathic effect when MT-2 cells were mixed with H9/HIV-1 cells. These substances also had no antiviral activity when added to cells, after virus challenge, at a time when binding and entry were complete. However, a high molecular weight (500,000) dextran sulphate blocked HIV-1 infection at both stages. Thus, the gp120-CD4 interactions mediating HIV-1 binding and HIV-1-induced syncytium formation are differentially affected by this class of polyanionic substances. Furthermore, size may be a determining factor in their potential application as anti-HIV treatment.

Plasma from two rhesus macaques (Macaca mulatta) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechallenge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complement-mediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency virus-positive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C12U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C12U) therapy and failed to produce IFN-α, IFN-β, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3−/− mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C12U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.

Background. Avian H5N1 influenza A virus is an emerging pathogen with the potential to cause substantial human morbidity and mortality. We evaluated the ability of currently licensed seasonal influenza vaccine to confer cross-protection against highly pathogenic H5N1 influenza virus in mice. Methods. BALB/c mice were inoculated 3 times, either intranasally or subcutaneously, with the trivalent inactivated influenza vaccine licensed in Japan for the 2005–2006 season. The vaccine included A/NewCaledonia/20/99 (H1N1), A/NewYork/55/2004 (H3N2), and B/Shanghai/361/2002 viral strains and was administered together with poly(I):poly(C12U) (Ampligen) as an adjuvant. At 14 days after the final inoculation, the inoculated mice were challenged with either the A/HongKong/483/97, the A/Vietnam/1194/04, or the A/Indonesia/6/05 strain of H5N1 influenza virus. Results. Compared with noninoculated mice, those inoculated intranasally manifested cross-reactivity of mucosal IgA and serum IgG with H5N1 virus, as well as both a reduced H5N1 virus titer in nasal-wash samples and increased survival, after challenge with H5N1 virus. Subcutaneous inoculation did not induce a cross-reactive IgA response and did not afford protection against H5N1 viral infection. Conclusions. Intranasal inoculation with annual influenza vaccine plus the Toll-like receptor—3 agonist, poly(I): poly(C12U), may overcome the problem of a limited supply of H5N1 virus vaccine by providing cross-protective mucosal immunity against H5N1 viruses with pandemic potential.

There is considerable controversy concerning the evidence for the presence of Chlamydia pneumoniae in the cerebrospinal fluid (CSF) of both multiple sclerosis (MS) patients and patients with other neurological diseases (OND). In order to clarify this issue, the laboratories at Vanderbilt University Medical Center (VUMC) and the University of South Florida (USF) examined the reproducibility of their respective PCR assays for the detection of C. pneumoniae DNA in the CSF of a common group of MS patients and OND controls. The two laboratories used different DNA extraction and PCR techniques in order to determine the prevalence of the C. pneumoniae genome in both monosymptomatic and clinically definite MS patients as well as in OND controls. In clinically definite MS patients, the VUMC and USF detection rates were 72 and 61%, respectively, and in patients with monosymptomatic MS, the VUMC and USF detection rates were 41 and 54%, respectively. The PCR signal was positive for 7% of the OND controls at VUMC and for 16% at USF. These studies confirm our previous reports concerning the high prevalence of C. pneumoniae in the CSF of MS patients. The presence of C. pneumoniae in patients with monosymptomatic MS would also suggest that infection with the organism occurs early in the course of the disease.

e14549 Background: Genetic aberrations such as SNPs and structural aberrations are associated with malignancies and cancer is regarded as a disease of the genome. Genome profiling of individual tumors is provided by high-throughput sequencing and is about to enter routine clinical practice with impact on treatment decisions and tumor classification (NIH [2014] The Cancer Genome Atlas). Rectal tumors show a great variation in response to preoperative chemoradiotherapy and, therefore, robust and easy to obtain individual biomarkers are needed in order to adjust treatment strategies for each patient. Methods: Copy-number profiles were generated for 13 rectal tumors (pre CRT; Stage II or higher) using shotgun sequencing (HiSeq). For a subset of the tumors (n=10) the 20 genes most commonly mutated in colon cancer were screened for mutations using the commercially available GeneRead DNAseq Colon Cancer Gene Panel (QIAGEN) with subsequent sequencing using an Illumina MySeq. Results: The copy-number profiles of t...

6072 Background: We selected HPV-associated laryngeal papillomas and squamous cell carcinomas for genomic analysis of viral induced benign and malignant neoplasms. The nearly universal presence of HPV in Recurrent Respiratory Papillomatosis (RRP) and in certain squamous cell carcinomas provides an important model for the study of biomarkers and clinical outcomes as well as the generation of robust panels of viral and genomic mutation biomarkers that can be used to follow disease progression. Methods: DNA was isolated from biopsies of three patients and sequenced on a NGS Illumina HiSeq platform. The sequences were compartmentalized in 250kbp bins, normalized, and compared to the mean (+/- 3SD) of Chronix controls to identify regions of chromosomal number imbalance (CNI). Sequences were also compared to databases with known viral genomes. Results: Sample 1: an exophytic, focally invasive, HPV positive (PCR), squamous cell carcinoma from a never-smoker. The full genome of HVP16 was detected in low amount (~...

Background: Considerable evidence suggests the role of an infectious agent in MS. The presence of Chlamydophila pneumoniae in CSF from patients with MS was shown earlier; to further examine this association the reactivity of the oligoclonal antibody response in the CSF of patients with MS to C pneumoniae antigens was determined and compared with other antigens. Methods: Seventeen patients with MS and 14 control subjects with other neurologic disease were studied. Affinity-driven immunoblot studies and solid-phase adsorption of CSF oligoclonal bands by elementary body antigens of C pneumoniae, viral antigens (measles and herpes simplex virus-1), bacterial antigen (Escherichia coli and Staphylococcus aureus), and heat shock protein-60 were performed.Results: Affinity-driven immunoblot studies demonstrated reactivity of oligoclonal bands in CSF samples from 16 patients with MS against elementary body antigens of C pneumoniae. None of the control subjects showed a prominent reactivity to elementary body antigens of C pneumoniae. In 14 of 17 patients with MS examined, oligoclonal bands were adsorbed either partially or completely from the CSF by elementary body antigens of C pneumoniae, but not by myelin basic protein, heat shock protein-60, or bacterial or viral antigens. In three patients with subacute sclerosing panencephalitis, adsorption of oligoclonal bands was seen with measles virus antigens but not with elementary body antigens of C pneumoniae.Conclusions: Oligoclonal bands in CSF of patients with MS include antibodies against Chlamydophila antigens.

Chlamydia pneumoniae has been demonstrated in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). Interferon-beta (IFN-beta) has favorable effects on the clinical course of MS. We investigated whether the beneficial effects of IFN-beta in MS may involve its role in regulating nitric oxide (NO) and interleukin-12 (IL-12) in macrophages, as these immune modulators form part of the innate immune response to intracellular pathogens, such as C. pneumoniae. Murine macrophages in cultures exposed to elementary body antigens or recombinant major outer membrane protein (rMOMP) of C. pneumoniae demonstrate a significant increase in NO as well as production of IL-12/p40 in culture supernatants compared with basal levels. Addition of murine IFN-beta increased NO activity in murine macrophages cultured with chlamydial antigens. Addition of neutralizing anti-IFN-beta antibody prevented the NO increase. In contrast to its effect on inducible NO synthase (iNOS), IFN-beta reduced induction of IL-12/p40 following culture with either elementary body antigens or rMOMP. Inhibition was reversed with anti-IFN-beta antibody. If C. pneumoniae infection is responsible for the inflammatory response in the pathogenesis of MS, the beneficial effects of IFN-beta in MS may be due to its enhancing intracellular NO activity while inhibiting secretion of the proinflammatory cytokine, IL-12.

Chlamydia Pneumoniae is not a known cause of skin infections, but unusual pathogens cause chronic infections in diabetic patients. Multiple idiopathic pyoderma gangrenosum–like (PG-like) lesions were refractory to multiple therapeutic agents in a diabetic patient who had C pneumoniae identified by serologic tests and polymerase chain reaction. Based on complete resolution by prolonged anti-chlamydial antibiotic therapy and concomitant decrease in serologic and titers determined by polymerase chain reactions, the PG-like lesions were presumed to be due to C pneumoniae . (J Am Acad Dermatol 2000;42:295-7.)

11060 Background: Recently we demonstrated the ability to detect the presence of invasive breast cancer by next generation sequence (NGS) analysis of cell-free DNA (cfDNA) in serum without prior knowledge of the specific gene perturbations of the neoplasm (J. Clin. Oncology 2010; 28 (15S):10505). We hypothesized that cfDNA levels would hold promise as a marker to monitor therapeutic efficacy in breast cancer. In this study, we sought to determine whether cfDNA decreases after surgery in patients with operable invasive breast cancer. Methods: Serum was collected before and 1-4 weeks after surgery from 16 breast cancer patients and also from 24 age and gender matched controls. cfDNA was isolated from the serum, amplified by Chronix proprietary method and sequenced on an Illumina HiSeq platform. The breast cancer derived cfDNA sequences from each patient were compartmentalized in 250kbp bins, normalized, and compared to the mean (+/- 3SD) of the controls to identify regions of chromosomal number imbalance (CNI). Genomic DNA was isolated from the primary tumor and blood WBC buffy coat, pre-amplified, sequenced and subjected to comparative analysis with cfDNA. Results: Analysis of pre- and post surgery serum demonstrate that CNIs are present in the pre-surgery serum cfDNA that correlated with the associated tumor CNI and was not observed in WBC genomic DNA. The number of tumor associated CNI DNA biomarkers ranged from as low as 3 to as high as 865. In 13 of 16 patients (81%) analyzed, the post-surgery cfDNA was free of detectable tumor related-CNI DNA. In the remaining 3 patients, only a partial reduction in tumor specific CNI DNA serum biomarkers was observed, since some markers were still detectable after surgery. Conclusions: cfDNA from primary breast carcinomas accurately reflects tumor CNI. Removal of the primary tumor results in the elimination of tumor cfDNA in the majority of patients. Thus cfDNA may be of clinical value in evaluation of therapeutic efficacy on a real time basis. Further work is needed to determine if residual tumor CNI in cfDNA after surgery is a marker of minimal residual disease which can be pursued as a prognostic marker.

5072 Background: Prostate cancer (PrCa) is the most frequent non-dermatological malignancy in the male population. Genomic instability resulting in copy number variation (CNV) is a hallmark of malignant transformation. CNV traces from tumors in cell-free DNA (cfDNA) of prostate cancer patients may be identified through massive parallel sequencing (MPS) of serum DNA. These CNV traces may be biomarkers of cancer with clinical applications for screening and follow-up. Methods: DNA was extracted from serum of 205 PrCa patients (Gleason 2 to10), 207 age matched male controls (HC), 10 men with benign hyperplasia (BPH) and 10 with prostatitis (PiS). DNA was amplified using random primers, tagged with a unique molecular identifier per sample, sequenced on a SOLiD system and aligned to the human genome (Build 37). Hits were counted in sliding 100kbp intervals and normalized. Using a random-resampling procedure, genomic regions showing copy number variations in cfDNA that distinguish PrCa from HC were selected. A model using 20 cfDNA regions was cross-validated and used as cfDNA biomarker. Receiver operator characteristics (ROC) curves were calculated for assessment of diagnostic performance by means of area under the curve (AUC). Results: To assess whether CNVs in cfDNA are indicative of PrCa, the number of regions with significant CNV deviation was counted in a first subset of 82 PrCa. Using only the number of regions as measure resulted in an AUC of 0.81 (0.7 – 0.9, p-7). This final model discriminated between PrCa and HC with an AUC of 0.92 (0.87 – 0.95) reaching a calculated accuracy of 83%. Both BPH and PiS could be distinguished from PrCa using the cfDNA CNV biomarker with a predicted accuracy of 90%. Conclusions: MPS revealed that only a limited number of chromosomal regions showing CNVs are necessary to achieve statistical separation between prostate cancer and controls. This technique may prove to be clinically useful for screening and follow up of men with prostate cancer.

11013 Background: Massive parallel sequencing provides high numbers of cell-free nucleic acid serum DNA sequences (cfDNA) that can detect trace amounts of tumor derived chromosomal imbalances and copy number variations (CNVs) in patients with cancer. The aim of this study was to determine if there is a difference between the cfDNA CNVs from patients with breast cancer (BrCa) compared to healthy controls. Methods: DNA extracted from serum samples of 225 BrCa (Stage 1 to 4) and 205 gender and age-matched healthy controls (HC) was amplified using random primers, tagged with a unique molecular identifier per sample, sequenced on an Illumina HiSeq system and aligned to the human genome (Build 37). Hits were counted in sliding 1Mbp interval regions and normalized. Using a Random-Resampling procedure, a model was established to distinguish BrCa from HC using the copy number variations (CNV) and cross validated. Results: From 1,100 rounds of random resampling (50/50), a set of 31 regions was selected, based on the frequency of occurrence in the models. Using 20 random sets of a 10-fold cross validation, the selected regions were found to be highly significant discriminators between BrCa and HC (p-5). When using a final linear model with 16 regions the AUC of a diagnostic ROC curve was found to be 0.895 for all samples, for Stage I and II the AUC was 0.86 compared to 0.93 for the higher stages. The final model included three regions from chromosome 8 and 1 and two regions from chromosome 15, the remaining regions were found as one per chromosome. Conclusions: Using comparative massive parallel sequencing of cfDNA from cancer patients vs. controls, we were able to show that a 16-region model based on CNV, is useful to distinguish patients with breast cancer from matched controls. Genomic instabilities that are shed into the circulation from breast cancer may play a role in screening, monitoring or as companion diagnostic tests in breast cancer.

Background: Expression of microbial protein sequences in eukaryotic cells transfected by transcriptional/translational permissive cDNA constructs can induce systemic humoral and cellular responses in vivo. Two methods of in vivo transfection have been described to date. One method uses large quantities of naked DNA injected into skeletal muscle. The second method uses relatively small quantities of DNA complexed to gold particles for bollistic penetration of the plasma membrane of keratinocytes. The major disadvantage of the bolistic method is that instrumentation is required which is not generally available. Objectives: The objectives of this study were to determine whether the use of DNA complexed with a cationic lipopolyamine could reduce the quantity of DNA required to induce systemic humoral responses following muscle transfection and whether similar DNA/lipopolyamine complexes could induce mucosal humoral responses following airway exposure. Study design: Balb/c mice were exposed by nasal aerosol or intramuscular inoculation to a mammalian transcriptional/translational permissive DNA construct containing the entire sequence for the HIV-1 envelope polyprotein. Experimental animals were further segregated by the number of exposures at 3-week intervals and whether the DNA was complexed to dioctadecylamidoglycylspermine (DOGS) at a 5:1 molar charge ratio of DOGS/DNA. Results: DOGS facilitated in vivo transfection of mouse muscle reduced the quantity of DNA required for a systemic humoral response to surface expressed HIV-envelope proteins by one order of magnitude. Exposure of airway mucosa to both 10 μg and 1 μg quantities of DNA complexed to DOGS produced systemic humoral responses to HIV-envelope as well as mucosal antibodies in pulmonary and colonic epithelia. Molecular modeling of DOGS/DNA complexes at the 5:1 charge ratio used in this study indicates that the DNA component is not exposed to aqueous solvents and may be relatively resistant to degradation under common biological environments. Conclusion: Facilitated transfer of DNA by DOGS to transcriptional/translational competent cells offers several distinct advantages to the use of DNA alone. Since significantly smaller amounts of DNA are required, the potential for the induction of antibodies against DNA itself lessens the likelihood for the development of a lupus-like syndrome. More importantly, however, is the apparent ability to transfect mucosal cells which results in the development of specific mucosal immune responses. This procedure may allow the development of general methods for the induction of mucosal immunity at the level of entry for mucosal pathogens without the disadvantages inherent in live attenuated vectors.

Objective: To demonstrate that antibodies against amino acids (aa) 603-622 of the SIV gp41 transmembrane glycoprotein enhance infection of SIV in vivo. Design: A synthetic peptide derived from aa 603-622 of SIVmac251 gp41 was synthesized and tested for immunogenicity in rabbits and SIV-infected rhesus macaques. Next, SIV-naive animals were immunized with either a recombinant vaccinia virus expressing the SIV gp160 envelope glycoprotein (VVrgp160) and boosted three times with aa 603-622 (group 1,four animals), wild-type vaccinia virus and boosted with aa 603-622 (group 2, two animals), or VVrgp160 followed by three doses of an irrelevant peptide (group 3, two animals). Animals were challenged with SIVmac251. Results: Peptide aa 603-622 was immunogenic in rabbits. SIV-infected rhesus monkeys immunized with the peptide developed two-three log increases in antibodies to this peptide and antibodies that could enhance SIV infection in vitro. SIV-naive rhesus macaques in group 1 had higher levels of antibody to the peptide by enzyme-linked immunosorbent assay and higher levels of enhancing antibodies at the time of SIV challenge than the animals in groups 2 or 3. Following challenge with SIVmac251 the group 1 animals had detectable p27 antigen longer than animals in group 2 and 3 and died of simian AIDS before the respective animals in the two control groups (P Conclusions: aa 603-622 of SIV gp41, like aa 579-613 of HIV gp41, can stimulate production of antibodies that enhance SIV and HIV infection in vitro. Furthermore, immunization with this peptide suppressed beneficial effects of a gp160 vaccine and appeared to enhance SIV infection in vivo.

The atomic structure of a truncated glycoprotein gp120 from human immunodeficiency virus 1 (HIV-1) that contains the principal neutralizing antigenic sites and the CD4 binding domain has been derived by molecular dynamics and calculation of potential energy using the DREIDING force field. The resultant N-glycosylated molecular model is consistent with known properties of gp120 and docks with CD4 with a substantial reduction in the sum of the internal potential energies of the individual proteins (delta E = -200 kcal/mol). The primary mechanism of recognition and binding is the insertion of the solvent-accessible Phe-43 of CD4 into a gp120 solvent-accessible acceptor pit formed by Trp-427, Tyr-435, and the high-mannose oligosaccharide N-linked to Asn-230. delta E for the nonglycosylated complex is reduced significantly (-75 kcal/mol). Binding is by pi-pi* interactions of the aromatic groups forming a hydrophobic, thermodynamically stable environment for these functional noncovalent bonding participants. This model for gp120 provides a theoretical basis for the evaluation of HIV molecular pathogenesis involving the env proteins, the analysis of conformation on functional immune response of the host, and the design of nonproteinaceous inhibitors specific for the CD4 binding site on gp120.

10505 Background: Next generation sequencing provides high numbers of circulating nucleic acid (CNA) serum DNA sequences that can detect trace amounts of these unique serum biomarkers in patients w...

The human interferon-gamma (IFN-gamma) receptor protein and mRNA were identified in the epidermoid carcinoma cell line A431 by radioligand binding and Northern hybridization, respectively. Receptor affinity and receptor number per cell were calculated by Scatchard analysis of saturation binding data. The half-life of the receptor mRNA was determined by actinomycin D inhibition of de novo transcription. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of covalently cross-linked 32P-labeled IFN-gamma and its receptor is consistent with a molecular mass of approximately 99 kD. Radioligand binding analysis revealed 27,000 high affinity (KD = 7.1 x 10(-10) M) receptors per cell. Northern hybridization using an IFN-gamma receptor cDNA probe revealed a single mRNA of 2.2 kb. The receptor mRNA half-life in actinomycin D-treated cells was 4.7 h.

Previous experiments had shown that two human monoclonal antibodies (huMAbs) directed against human immunodeficiency virus type 1 (HIV-1) enhanced HIV-1 infection in vitro (Robinson et al., Proc. Natl. Acad. Sci. USA, 87:3185-3189, 1990). This complement-mediated, antibody-dependent enhancement (C'-ADE) of HIV-1 infection caused 12-fold increases in reverse transcriptase released from MT-2 cells. In the study reported here, it was demonstrated that both of these huMAbs, 86 and V10-9, bound to an immunodominant peptide in gp41 (amino acids 586 to 620). This peptide blocked C'-ADE of HIV-1 infection in vitro regardless of whether huMAb 86 or human polyclonal anti-HIV was used as the source of anti-HIV antibody. Blockade of enhanced infections was characterized by decreases in antigen synthesis, cytopathic effect, and reverse transcriptase release. The ability of the huMAbs to enhance infection was determined to be dependent upon specific peptide reactivity and not dependent upon immunoglobulin subclass, complement fixation, or gross antigen reactivity. Since the peptide to which enhancing antibodies bind is immunodominant and does not bind neutralizing antibodies, it may be worthwhile to investigate deletion of this 35-amino-acid peptide from candidate anti-HIV vaccines.

In this study it is demonstrated that complement-mediated antibody-dependent enhancement (C'-ADE) of HIV-1 infection in vitro is blocked by murine monoclonal antibodies to CD4 and complement receptor type 2 (CR2) while HIV-1 infection in the absence of C'-ADE is blocked by anti-CD4 but not anti-CR2 monoclonal antibodies. The anti-CR2 murine monoclonal antibody, OKB7, blocked C'-ADE of HIV-1 infection at concentrations greater than 1 microgram/ml. The anti-CD4 monoclonal antibody, OKT4a, but not OKT4f blocked C'-ADE at concentrations greater than 0.06 microgram/ml. HIV-1 infections were quantitated by cytopathic effect, indirect immunofluorescence, and reverse transcriptase release. It appears from these in vitro studies that C'-ADE of HIV-1 infection requires both CD4 and complement receptors while HIV-1 infection in the absence of antibody and complement requires only CD4.

Over the past year significant progress has been made in mapping those regions of the HIV-1 envelope glycoproteins involved in virus neutralization and virus enhancement. These functional, antigenic domains of the gp160 are illustrated in Fig. 1. The role of neutralization in vaccine development is still unresolved, although high-titer antibody to the V3 loop of HIV-1 appears to be correlated with the ability to prevent HIV-1 infection by the homologous strain in chimpanzees. Therefore, the mechanism of type-specific neutralization of HIV appears to be clearly defined. The problem of group-specific neutralization of HIV-1 is still a mystery. Nevertheless, the finding that secondary structure is important for the generation of group-specific neutralizing mAbs and that carbohydrate is an important determinant for group-specific neutralization suggests that non-linear determinants are important. Answers to the question of group-specific neutralization should be available in the next few years. The role of HIV-1-enhancing antibodies in pathogenesis is not well understood. Nevertheless, the ability of patient serum to only enhance the patient's own isolate and not neutralize that isolate suggests that enhancing antibodies are important. Furthermore, the findings that enhancing antibody titers increase in SIV infection and peak immediately prior to the death of the animal suggests that such antibodies may play a role in SIV pathogenesis. With the identification and domain mapping of enhancing hu-mAbs, it should be possible to evaluate directly the role of enhancing domains in HIV and SIV pathogenesis by challenging animals in the presence of pure enhancing antibody. Only when these experiments are performed will it be possible to evaluate what role, if any, enhancing antibodies play in HIV pathogenesis. The above questions, when answered, are likely to provide important insights into lentivirus pathogenesis and help researchers to produce a safe and effective anti-HIV vaccine.

In crude homogenates of the submaxillary gland of the male mouse, epidermal growth factor activity is found almost entirely in a high molecular weight complex. The complex has a molecular weight of about 74,000 and appears to be composed of 2 molecules of epidermal growth factor (6,045 molecular weight) and 2 molecules of a binding protein (29,300 molecular weight). The complex is stable only in the range of pH 5.0 to 8.0, and its behavior during isoelectric focusing, gel filtration, and ultracentrifugation suggests that it may be in a slow equilibrium with its subunits. In addition, the complex appears to undergo a concentration-dependent aggregation. The biological activity of the high molecular weight complex is proportional to its fractional content of the low molecular weight factor. No evidence was found which indicated that the binding protein might affect the biological activity of the low molecular weight epidermal growth factor. It is postulated that the high molecular weight form of epidermal growth factor is an enzyme-product complex. The substrate is presumed to be the epidermal growth factor precursor which is recognized by a specific arginine esterase, the binding protein.

Modulation of the intracellular concentration of cyclic AMP has been associated with a regulatory role in cell division, cell morphology, and physical properties of the plasma membrane. Untransformed rat kidney cells in culture exhibit epitheloid morphology, high intracellular cyclic AMP levels, and contact inhibition of growth. Untransformed rat kidney cells transformed with the Kirsten murine sarcoma virus exhibit a low cyclic AMP content, rapid growth rate, and a loss of contact inhibition. Scanning electron microscopy reveals a distinctive difference in the surface structure of the two cell types during Gl of the cell cycle. The surface of the transformed cell is covered with microvilli while its untransformed counterpart is devoid of microvilli. The presence of microvilli can be controlled as a function of temperature by two temperature-sensitive mutants of the Kirsten sarcoma virus (ts6t6 and ts371 cl 5). In the ts6t6 mutant, growth at 32 degrees C results in a low cyclic AMP content and the presence of microville, while growth at 39 degrees C results in a high cyclic AMP content and a decrease in microvilli. The opposite effect is seen with the ts371 cl 5 mutant. Correlation of cyclic AMP content with the presence of microvilli suggests that this surface phenomenon is a function of cyclic AMP concentration.

Chemically and enzymatically synthesizeddiastereomeric 2′,5′- phosphorothioate dimer, trimer and tetramer cores and their 5′ - mono- and triphosphates demonstrate marked differences in their ability to bind to and activate RNase L from L929 cell extracts in radiobinding, core-cellulose and rRNA cleavage assays1,2 (Fig. 1). These are the first 2-5A cores that are able to bind to and activate Mase L. The enzymatically synthesized 2′,5′- phosphorothioate dimer and trimer 5′-triphosphates can also bind to and activate mase L1.

Nine phenolic chelating agents were examined as antidotes for acute uranyl acetate intoxication. The ability of these compounds to promote survival was determined and the ability of two of them to remove uranium from the liver and kidney was compared with that of Na3 CaDTPA. The most effective compounds of this type in promoting survival were found to be Tiron and p‐aminosalicylic acid, but Tiron was much more effective in decreasing the uranium burden of the liver and kidneys. These compounds exhibit an antidotal action at higher uranium levels than the compounds reported in earlier studies. The compounds all seem to possess a very modest toxicity when administered ip.

Although thiones (R2C = S) such as methimazole (tautomer) and thioacetamide have been shown in animal studies to be weak carcinogens (Gupta, 1955, 1956; Jemec, 1977; Stall et al., 1978), they fall to be detected as mutagens for Salmonella typhimurium by the standard Ames assay protocol (McCann et al., 1975). In Drosophila, however, thioacetamide has been reported to increase significantly the number of sex-linked lethal mutations (Magnusson and Ramel, 1978) while methimazole produces dominant male mutations in mice (Akatsuka et al., 1979). Since the metabolic oxidation product of thioacetamide, thioacetamide S-oxide, has been demonstrated to be a more rapid and efficient hepatic necrotoxin (Hunter et al., 1977; Porter et al., 1979), the failure of the Ames assay to detect mutagenic thiones may be secondary to the mechanics of metabolic activation to S-oxides. In this study, we have shown that thioacetamide Soxide is mutagenic in the Ames assay without metabolic activation by mammalian microsomal ($9) additives and that such additives block the observed mutagenicity of the S-oxide.

The acquired immunodeficiency syndrome (AIDS) is characterized by severe unrelenting opportunistic infections and/or Kaposi's sarcoma associated with a dysfunction of cell mediated immune responses. In addition to cutaneous anergy and inversion of T-helper/inducer to T-suppressor cell ratios usually observed in AIDS, we have observed in three AIDS cases with multiple opportunistic infections that spontaneous and interferon (IFN) stimulated natural killer (NK) cell activity against K-562 cells is absent or severely depressed as compared to controls. Spontaneous and IFN stimulated NK cell activity in promiscuous male homosexual controls was similar to that observed in heterosexual controls. The control subjects had T-helper:T-suppressor ratios ranging from 1.1 to 2.4 in contrast to the AIDS subjects who had typical inverted ratios of T-cells ranging from 0.17 to 0.54. HNK-1, a monoclonal antibody that identifies NK and antibody-dependent cytotoxic cells, marked 3-22% of peripheral blood lymphocytes from AIDS patients in comparison to 8-38% of lymphocytes from controls. A larger series will be required in order to ascertain the true incidence of NK cell anergy in AIDS and whether or not subgroups exist based on the association of Kaposi's sarcoma in the disease process.

Tropocollagen is derived from an extracellular precursor, procollagen. Conversion to tropocollagen is accomplished by one or more tissue pioteases dependent in vitro on the presence of serum in the culture medium. Twenty-four hour cultures in which serum has been excluded yield an apparently undegraded precursor, procollagen I. The latter is approximately twice the size of tropocollagen, possesses an acidic pl in contrast to the alkaline pl of tropocollagen, and shares secondary structural characteristics common to tropocollagen. Procollagen I exhibits a sharp thermal transition point at 39° with a ΔT of 2° indicating that the collagenous portion of the molecule is in the triple helical configuration prior to proteolytic excision from the parent molecule. The amino acid composition is remarkable when compared to tropocollagen in the large quantity of acidic residues, decreased glycine and imino acids, and the presence of cystcine. Three models of procollagen I structure are presented and discussed relative to the available experimental evidence.

The major internal structural protein (p30 gag ) of the Moloney leukemia virus and the endogenous Y-1 murine oncornavirus was examined for biochemical and biophysical manifestations of interstrain antigenic variation. Although the two viral proteins share murine group-specific antigenic determinants, the Y-1 virus p30 appeared to have both a lower relative number of such determinants and a decreased affinity at the cross-reactive sites for Moloney virus p30 monospecific antibodies. Further, immunological analysis indicated the presence of unique antigenic sites on the Moloney virus p30 not shared by the analogous Y-1 virus molecule. The two polypeptides copurified and had similar isoelectric points (pH 6.2 to 6.3) and sedimentation coefficients (2.47S). However, equilibrium sedimentation yielded a significant mass difference between the two proteins, 28,300 ± 600 and 31,000 ± 300 daltons for the Moloney and Y-1 virus molecules, respectively. Amino acid analysis indicated a concomitant increase in total residues for the Y-1 virus p30, although a number of residues appeared to have been conserved between the two viral proteins. Conformational studies and hydrodynamic calculations demonstrated marked secondary and tertiary structural differences; with the Y-1 virus p30 being an asymmetric prolate ellipsoid containing 27 to 28% α-helix and Moloney virus p30 being somewhat more spherical and possessing an α-helical content of 50 to 55%. Two-dimensional mapping of 125 I-labeled tryptic peptides of each p30 suggested that considerable sequence heterogeneity is responsible for many of the biophysical, biochemical, and immunochemical differences in these two analogous structural proteins.

Based on recent reports of antibody-dependent enhancement of human immunodeficiency virus type 1 (HIV-1) infection in vitro by serum from HIV-1-infected humans, sera from HIV-1 antibody-positive chimpanzees (Pan troglodytes) was evaluated for enhancing activity in an in vitro infection assay that uses MT-2 cells (a human lymphoblastoid cell line). Although fresh chimpanzee serum was found to have pronounced infection-enhancing properties in the absence of antibody to HIV-1, this effect was abolished by heat inactivation (57 degrees C, 1 hr) or treatment with cobra venom anticomplementary protein. Heat-inactivated, HIV-1 antibody-positive chimpanzee serum could enhance HIV-1 infection of MT-2 cells in vitro when combined with fresh, normal human serum. By serial serum samples from three HIV-1-infected chimpanzees, HIV-1 antibody-positive chimpanzees are shown to develop enhancing antibodies early in infection (2 mo postchallenge), whereas neutralizing antibodies develop later. Over the course of HIV-1 infection, this enhancing activity decreases while neutralizing activity increases, suggesting a possible role for enhancing and neutralizing activities in HIV-1 pathogenesis. The enhancing activity of an IgG fraction used to passively immunize chimpanzees against HIV-1 infection is shown to be present at dilutions as high as 1:65,000, offering an interesting possible reason for the failure of passive immunization to protect chimpanzees from HIV infection. These results suggest that serum from HIV-1-immunized chimpanzees might be tested to determine whether current HIV-1 candidate vaccines induce production of antibodies that mediate antibody-dependent enhancement of HIV-1 infection in this in vitro assay.

Procollagen from the culture medium of human foreskin fibroblasts is efficiently adsorbed on controlled-pore glass (CPG) beads. Elution of adsorbed protease(s), capable of procollagen degradation, is accomplished with 1 m phosphate. This allows subsequent purification steps to be accomplished without detectable degradation of the high-molecular-weight procollagen form which is subsequently eluted with 1 m Tris. Analysis of the Tris elution fraction from CPG beads by sodium dodecyl sulfate-agarose-polyacrylamide electrophoresis on 2% gels indicated that the majority of protein is types I and III procollagens and partially processed intermediates. Types I and III procollagens were separated by DEAE-cellulose chromatography, and presumptive undegraded type I procollagen was resolved from processed forms by molecular sieve chromatography in 1 m CaCl2 on agarose beads. The high-molecular-weight type I human procollagen isolated by this method was found to contain both amino and carboxy-terminal propeptides. Two α1 and one α2 procollagen chains, disulfide bonded via the carboxy-terminal propeptides, are present per molecule. This procedure represents an efficient and relatively rapid method for preparing human procollagen in sufficient quantity for detailed chemical analysis.

Human immunodeficiency virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS), contains two heavily glycosylated envelope proteins, gp120 and gp41, which mediate attachment of virions to glycosylated cell surface receptor molecules (CD4 antigens) and appear to be responsible for syncytium formation and associated cytopathic effects of this virus. A comprehensive study of the effects of N-linked glycoprotein processing inhibitors on HIV-1 replication, infectivity, cytopathicity, target-cell infectibility, syncytium formation, and gp120 electrophoretic mobility was conducted to assess the importance of protein glycosylation in the pathogenesis of HIV-1 in vitro. The electrophoretic mobility of gp120 was decreased when gp120 was synthesized in the presence of castanospermine or 1-deoxynojirimycin (inhibitors of glucosidase I), increased when gp120 was synthesized in the presence of 1-deoxymannojirimycin (mannosidase I) or swainsonine (mannosidase II), and unaffected when gp120 was synthesized in the presence of bromoconduritol (glucosidase II). Inhibition by tunicamycin (lipid-linked oligosaccharide precursor synthesis), castanospermine, 1-deoxynojirimycin, and 1-deoxymannojirimycin attenuated HIV-1 infectivity and blocked HIV-1-induced syncytium formation and cytopathicity, whereas bromoconduritol and swainsonine failed to have such effects. None of the inhibitors interfered with virus replication in acutely infected cells or affected the ability of target cells to form syncytia with untreated HIV-1-infected cells. These results demonstrate that protein N-glycosylation is critical to the pathogenesis of HIV-1 at the levels of viral infectivity and cytopathicity but not at the level of virus replication or of host-cell infectibility.

Physical, chemical, and enzymatic properties of the protein which binds epidermal growth factor (EGF) have been examined. A molecular weight of 29,300 was determined from sedimentation equilibrium studies, in agreement with a value of 30,000 calculated from the s020, w (2.83 S) and D020, w (8.73 x 10-7 cm2 s-1). It has an E1%1 cm at 280 nm of 15.6 and the isoelectric point is 5.6. The circular dichroic spectrum indicates a major nonhelical character with a minor amount of α helical structure. The amino acid composition was determined. No free sulfhydryl groups were detected. Hexosamine analysis revealed the presence of glucosamine and galactosamine. EGF-binding protein was found to be an arginine esterase with a restricted substrate specificity. Kinetic studies of the catalytic hydrolysis of Nα-benzoyl-l-arginine ethyl ester revealed a Km of 130 µm and a pH optimum in the range of pH 7.8 to 8.5, where the specific activity is approximately 390 µmoles per min per mg. This arginine esterase is not identical, but is related antigenically, to the arginine esterases associated with the nerve growth factor complex. The EGF-binding protein is postulated to function in the enzymatic liberation of active EGF from an inactive precursor.

Studies on a rat sarcoma link plasma membrane alterations to the activity of DNA-directed RNA polymerase II.

A rapid, quantitative, and nonsubjective method of interferon assay is described, which can be readily applied to clinical specimens. Automated data acquisition and data reduction allowed a significant increase in volume per unit of time over existing methodologies. Plasma always yielded higher (usually 2:1) interferon values than did serum obtained simultaneously. Ranges of interferon levels in plasma in normal control populations are reported as well as ranges for clinical virology laboratory technicians and patients with terminal malignancies or collagen vascular diseases.

Amides of thiophosphoric acid were studied, in order to initiate correlations of mutagenicity with structure, by use of a modified Ames assay. Representative thiophosphoramides of the structure (XArNH)3PS were not mutagenic, irrespective of whether X was a reference hydrogen atom, an electron-withdrawing, or an electron-donating group. One phosphoramidothioate of the structure (2,4-X2ArNH)P(S)(OCH2CH3)2 effected base-pair mutation when X was F (but not when X was CH3), when S-9 liver homogenate with exogenous NADP was used; when X was H, only inconsistent mutagenic activity following metabolic activation was observed even at concentrations near those that produced acute cellular toxicity. Mutagenicity of these N-arylamides thus appears to follow guidelines concluded for esters, (RO)3PS, i.e. that mutagenesis is most probable when two groups are small enough to permit nucleophilic attack by a biomacromolecule on the electrophilic phosphorus atom. That the third group should be electron withdrawing...

10 patients with the acquired immunodeficiency syndrome (AIDS), AIDS-related complex (ARC), or lymphadenopathy syndrome (LAS) were given 200-250 mg ampligen, a mismatched double-stranded (ds) RNA with in-vitro antiviral activity against human immunodeficiency virus (HIV), twice a week for up to 18 weeks, without side-effects or toxicity. In all 9 patients who were positive for HIV RNA in peripheral blood mononuclear cells before therapy, levels became undetectable between days 10 and 40 of the start of therapy. 6 of the 7 patients with ARC or LAS also showed a progressive reduction in HIV load as measured by co-culture assays. All 10 patients had augmentation of delayed-type hypersensitivity skin reactions. Other changes noted during ampligen therapy included an increase in or maintenance of numbers of helper-inducer T lymphocytes, improvements in HIV-related symptoms, rises in titre of neutralising antibodies against HIV, and restoration of proper functioning of the natural lymphocyte antiviral dsRNA-dependent (2'-5'-oligoadenylate/RNA-ase L) pathway. Thus, in the short term, ampligen seems to have the dual ability to restore immunological function and to control HIV replication.

Tris (1-aziridinyl) phosphine oxide (TEPA) and tris (1-aziridinyl) phosphine sulfide (thio-TEPA) induced base pair mutations in the Ames mutagenic assay. Thio-TEPA required metabolic activation while TEPA was active without metabolic activation. Growth of a human vaginal carcinoma (A431), a human breast carcinoma (MDA-MB-231), and a human cervical carcinoma (HeLa) were inhibited in soft agar in vitro at concentrations which induced mutagenesis in the Ames Assay. A fourth line, JEG choriocarcinoma, was sensitive to the antigrowth properties of both drugs at concentrations below that which induced mutagenesis. These data suggest that as more antineoplastic agents become available, and as mean survival times increase, knowledge of the relative in vitro sensitivity of a patient's neoplasm to a specific antineoplastic drug (i.e., dose required for growth inhibition) as a function of its mutagenic index might be useful for prediction of clinical remission, as well as the risk of secondary neoplasm induction.

A messenger RNA fraction isolated from cultured Chinese hamster lung (CHL) cells programs in a cell-free system prepared from wheat germ the efficient incorporation of [ 14 C] proline into newly synthesized protein with a significant fraction of the incorporated substrate being digestible with bacterial collagenase. This reaction requires both subcellular fractions, an energy source, and is inhibited by the antibiotic puromycin. The relative amount of collagenase-digestible to non-digestible cell-free product depends upon the ratio of CHL mRNA to wheat germ lysate, is not affected by either the Mg 2+ or K + concentrations employed, and under optimal condition, approximately 38% of the total incorporated substrate is collagenase-sensitive. Electrophoresis on SDS-polyacrylamide gels of the products programmed by CHL mRNA indicates that the collagenase-digestible material corresponds in size to a procollagen chain with an apparent molecular mass of approximately 170,000 daltons. These studies suggest that the collagen α1 (V) chain is initially synthesized as a precursor procollagen chain and demonstrate that a significant amount of the mRNA in Chinese hamster lung cells codes for this protein.

Cytolytic activity of human mononuclear peripheral blood leukocytes from healthy donors, cultured in interleukin-2 conditioned medium, was abrogated by in vitro infection with the lymphadenopathy associated virus (LAV) isolate of the human immunodeficiency virus (HIV). Although viral antigens are not expressed in cultured cells until 14 days postinfection, cytolytic activity was lost as early as 3 days after infection. Loss of cytolytic function was not a result of the release of suppressive factors from either infected cells or uninfected CEM cells since supernatants from neither infected cultures nor CEM cell cultures had any inhibitory effects on the function of uninfected cells. Cultured lymphocytes expressing Leu 11b were also shown to express HIV antigens via immunofluorescence after 14 days in culture. These results suggest that natural killer (NK) cells, as defined by expression of Leu 11b, were infected by HIV in vitro and the loss of lytic function was likely a direct consequence of that infection.

The recent discovery that HTLV-I infected T-cells have an enhanced susceptibility to HTLV-III/LAV infection with concurrent cell lysis ( S. Harada, Y. Koyanagi, and N. Yamamoto (1985) Science, 229, 563–566) led us to investigate a possible role for HTLV-II infected T cells in this respect. HTLV-II-bearing C3 cells were exposed to HTLV-III/LAV and the subsequent infection monitored by indirect immunofluorescence (IIF) of cells, reverse transcriptase (RT) activity in culture fluids, and hemacytometer cell counts. HTLVIII/LAV antigen expression was detected within 2 days whereas RT activity was detected within 3 days postinfection. The presence of immunofluorescent positive cells and RT activity was accompanied by cell lysis. Double infection of C3 cells was confirmed by IIF using a goat polyclonal antibody directed against the core p24 protein for detecting HTLVII, and high titer serum from a pre-AIDS patient for detecting HTLV-III/LAV. We conclude that HTLV-I and HTLV-II infected T-cells share an enhanced susceptibility to HTLVIII/LAV infection with the promotion of cell lysis.

The proteolytic enzyme, clostridiopeptidase B (clostripain), from Clostridium histolyticum culture filtrates has been purified to apparent homogeneity as judged by ultracentrifugation, electrophoresis in polyacrylamide gels, immunodiffusion, and immunoelectrophoresis. The enzyme has an apparent molecular weight of 50,000 from sedimentation equilibrium studies. Optical rotatory dispersion studies suggest a low α-helical content. Clostridiopeptidase B exhibits proteolytic as well as amidase-esterase properties. The specificity is confined primarily to arginine residues, although hydrolysis proceeds to a minor degree in most lysine-containing substrates.