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Supplementary Data from Next Generation Sequencing of Serum Circulating Nucleic Acids from Patients with Invasive Ductal Breast Cancer Reveals Differences to Healthy and Nonmalignant Controls

Circulating nucleic acids (CNA) isolated from serum or plasma are increasingly recognized as biomarkers for cancers. Recently developed next generation sequencing provides high numbers of DNA sequences to detect the trace amounts of unique serum biomarkers associated with breast carcinoma. Serum CNA of 38 women with ductal carcinoma was extracted and sequenced on a 454/Roche high-throughput GS-FLX platform and compared with healthy controls and patients with other medical conditions. Repetitive elements present in CNA were detected and classified, and each repetitive element was normalized based on total sequence count or repeat count. Multivariate regression models were calculated using an information-theoretical approach and multimodel inference. A total of 423,150 and 953,545 sequences for the cancer patients and controls, respectively, were obtained. Data from 26 patients with stages II to IV tumors and from 67 apparently healthy female controls were used as the training data set. Using a bootstrap method to avoid sampling bias, a five-parameter model was developed. When this model was applied to a validation data set consisting of patients with tumor stage I (n = 10) compared with healthy and nonmalignant disease controls (n = 87; 1,261,561 sequences) a sensitivity of 70% at a specificity of 100% was obtained. At a diagnostic specificity level of 95%, a sensitivity of 90% was calculated. Identification of specific breast cancer–related CNA sequences provides the basis for the development of a serum-based routine laboratory test for breast cancer screening and monitoring. Mol Cancer Res; 8(3); 335–42

Supplementary Table 3

Supplementary Tables, Supplementary Figure Legend and Supplementary Figures SUPPLEMENTARY TABLE,1:DISCOVERY COHORT (n=23) SUPPLEMENTARY TABLE,2:VALIDATION COHORT (n=33)

Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum–derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers.Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed.Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs.Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074–81. ©2017 AACR.

Coronary sinus ostial obstruction is an exceedingly rare anomaly that is particularly important to diagnose in patients with single-ventricle heart disease before surgical palliation. We present 2 cases, an infant and an adult, diagnosed with coronary sinus ostial obstruction, with different clinical outcomes due to timing of diagnosis. (Level of Difficulty: Intermediate.), Central Illustration

To explore the effect of lingual artery ligation on tongue vascularity, we performed an analysis of 25 patients who underwent transoral robotic surgery for base of tongue cancers (May 2011 to December 2019). Hounsfield units of the intrinsic muscles (IMs) and genioglossus muscles (GGs) were measured in postoperative imaging (mean 4 months) as a surrogate for vascularity. In ligated patients (n = 15), the values from the ligated/resected side of the tongue were compared with the contralateral side and the nonligated side of resection. Individually, IMs and GGs on the ligated side demonstrated no significant difference to the contralateral side (P = .662 and .618, respectively). Ligation produced a significant decrease in IM measurements but no difference between GG values vs nonligated patients (P = .050 and .818, respectively). No difference was appreciated in mean values for combined IMs and GGs between cohorts (P = .212). No gross tongue atrophy or complications were incurred. Future studies are warranted to delineate long-term effects.

BackgroundRintatolimod is a selective TLR3 agonist, which has demonstrated clinical activity for ME/CFS in Phase II and Phase III double-blind, placebo-controlled, randomized, multi-site clinical trials.Methods and findingsA hypothesis-based post-hoc analysis of the Intent to Treat (ITT) population diagnosed with ME/CFS from 12 independent clinical sites of a Phase III trial was performed to evaluate the effect of rintatolimod therapy based on disease duration. The clinical activity of rintatolimod was evaluated by exercise treadmill tolerance (ETT) using a modified Bruce protocol. The ITT population (n = 208) was divided into two subsets of symptom duration. Patients with symptom duration of 2-8 years were identified as the Target Subset (n = 75); the remainder (8 year) were identified as the Non-Target Subset (n = 133). Placebo-adjusted percentage improvements in exercise duration and the vertical rise for the Target Subset (n = 75) were more than twice that of the ITT population. The Non-Target Subset (n = 133) failed to show any clinically significant ETT response to rintatolimod when compared to placebo. Within the Target Subset, 51.2% of rintatolimod-treated patients improved their exercise duration by ≥25% (p = 0.003) despite reduced statistical power from division of the original ITT population into two subsets.Conclusion/significanceAnalysis of ETT from a Phase III trial has identified within the ITT population, a subset of ME/CFS patients with ≥2 fold increased exercise response to rintatolimod. Substantial improvement in physical performance was seen for the majority (51.2%) of these severely debilitated patients who improved exercise duration by ≥25%. This magnitude of exercise improvement was associated with clinically significant enhancements in quality of life. The data indicate that ME/CFS patients have a relatively short disease duration window (Trial registrationClinicalTrials.gov: NCT00215800.

Visual deficits after traumatic brain injury (TBI) are common, but interventions that limit the post-trauma impairments have not been identified. We have found that treatment with the cannabinoid type-2 receptor (CB2) inverse agonist SMM-189 for 2 weeks after closed-head blast TBI greatly attenuates the visual deficits and retinal pathology this otherwise produces in mice, by modulating the deleterious role of microglia in the injury process after trauma. SMM-189, however, has not yet been approved for human use. Raloxifene is an FDA-approved estrogen receptor drug that is used to treat osteoporosis, but it was recently found to also show noteworthy CB2 inverse agonism. In the current studies, we found that a high pressure air blast in the absence of raloxifene treatment yields deficits in visual acuity and contrast sensitivity, reductions in the A-wave and B-wave of the scotopic electroretinogram (ERG), light aversion, and increased pupil constriction to light. Raloxifene delivered daily for two weeks after blast at 5–10 mg/kg mitigates or eliminates these abnormalities (with the higher dose generally more effective). This functional rescue with raloxifene is accompanied by a biasing of microglia from the harmful M1 to the helpful M2 state, and reductions in retinal, optic nerve, and oculomotor nucleus pathology. We also found that raloxifene treatment is still effective even when delayed until 48 h after TBI, and that raloxifene benefit appears attributable to its CB2 inverse agonism rather than its estrogenic actions. Our studies show raloxifene is effective in treating visual injury after brain and/or eye trauma, and they provide basis for phase-2 efficacy testing in human clinical trials.

Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum–derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers.Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed.Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs.Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074–81. ©2017 AACR.

Toll-like receptors (TLRs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. These essential immune responses are triggered by common TLR pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal Toll/IL-1 domain. Toll/IL-1 adapter protein cascades are induced by an activated Toll/IL-1 to induce transient transcription responses. All TLRs, with the exception of TLR3, use an MyD88 adapter to Toll/IL-1 to initiate a proinflammatory cascade. TLR3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded RNA agonists. This non-MyD88 pathway induces both NF-κB and type 1 interferon responses. By using a TLR3-restricted double-stranded RNA agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. The mechanism of this differential response is consistent with a relative down-regulation of the NF-κB inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. Our findings suggest evaluation of TLR3 agonists in drug therapy.

Chronic fatigue syndrome/ Myalgic encephalomyelitis (CFS/ME) is a poorly understood seriously debilitating disorder in which disabling fatigue is an universal symptom in combination with a variety of variable symptoms. The only drug in advanced clinical development is rintatolimod, a mismatched double stranded polymer of RNA (dsRNA). Rintatolimod is a restricted Toll-Like Receptor 3 (TLR3) agonist lacking activation of other primary cellular inducers of innate immunity (e.g.- cytosolic helicases). Rintatolimod also activates interferon induced proteins that require dsRNA for activity (e.g.- 2ʹ-5ʹ adenylate synthetase, protein kinase R). Rintatolimod has achieved statistically significant improvements in primary endpoints in Phase II and Phase III double-blind, randomized, placebo-controlled clinical trials with a generally well tolerated safety profile and supported by open-label trials in the United States and Europe. The chemistry, mechanism of action, clinical trial data, and current regulatory status of rintatolimod for CFS/ME including current evidence for etiology of the syndrome are reviewed.

Using recently available mass sequencing and assembly technologies, we have been able to identify and quantify unique cell-free DNA motifs in the blood of patients with multiple sclerosis (MS). The most common MS clinical syndrome, relapsing-remitting MS (RRMS), is accompanied by a unique fingerprint of both inter- and intragenic cell-free circulating nucleic acids as specific DNA sequences that provide significant clinical sensitivity and specificity. Coding genes that are differentially represented in MS serum encode cytoskeletal proteins, brain-expressed regulators of growth, and receptors involved in nervous system signal transduction. Although coding genes distinguish RRMS and its clinical activity, several repeat sequences, such as the L1M family of LINE elements, are consistently different in all MS patients and clinical status versus the normal database. These data demonstrate that DNA motifs observed in serum are characteristic of RRMS and disease activity and are promising as a clinical tool in monitoring patient responses to treatment modalities.

Circulating nucleic acids (CNA) isolated from serum or plasma are increasingly recognized as biomarkers for cancers. Recently developed next generation sequencing provides high numbers of DNA sequences to detect the trace amounts of unique serum biomarkers associated with breast carcinoma. Serum CNA of 38 women with ductal carcinoma was extracted and sequenced on a 454/Roche high-throughput GS-FLX platform and compared with healthy controls and patients with other medical conditions. Repetitive elements present in CNA were detected and classified, and each repetitive element was normalized based on total sequence count or repeat count. Multivariate regression models were calculated using an information-theoretical approach and multimodel inference. A total of 423,150 and 953,545 sequences for the cancer patients and controls, respectively, were obtained. Data from 26 patients with stages II to IV tumors and from 67 apparently healthy female controls were used as the training data set. Using a bootstrap method to avoid sampling bias, a five-parameter model was developed. When this model was applied to a validation data set consisting of patients with tumor stage I (n = 10) compared with healthy and nonmalignant disease controls (n = 87; 1,261,561 sequences) a sensitivity of 70% at a specificity of 100% was obtained. At a diagnostic specificity level of 95%, a sensitivity of 90% was calculated. Identification of specific breast cancer–related CNA sequences provides the basis for the development of a serum-based routine laboratory test for breast cancer screening and monitoring. Mol Cancer Res; 8(3); 335–42

Background: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a debilitating disease of unknown pathogenesis consisting of a variety of flu-like symptoms including severe fatigue. Initial analysis of the use of rintatolimod (Poly I: Poly C 12U), a selective TLR3 agonist, in a Phase III, double-blind, randomized, placebo-controlled trial of CFS/ME demonstrated statistical significance (p 9 minutes. Analysis of improvement in exercise performance at the ≥ 25% and ≥ 50% levels using ET at 40 weeks compared to baseline was performed for the intent-to-treat (ITT) population (n=208) using the pre-specified baseline exercise stratum (baseline ET duration >9 minutes). For this subset of patients (n=126), 33% (n=20), and 12% (n=8) of rintatolimod vs. placebo patients, respectively, improved ET duration by ≥ 25% (p=0.004) while 23% (n=14) compared to 4.5% (n=3) of rintatolimod vs. placebo patients, respectively improved ET duration by ≥ 50% (p=0.003). This corresponds to increases of ≥ 186 and ≥ 373 seconds for patients receiving rintatolimod, respectively, at ≥ 25% and ≥ 50% improvement responses. A frequency distribution analysis of ≥ 25% improvement, 9 minutes cohort showed net improvement to be 18.3% for the rintatolimod cohort vs. 4.6% deterioration for placebo (p=0.015). A continuous responder analysis using 5% increments from ≥ 25% to ≥ 50% provided a robust clinical enhancement in ET effect in the rintatolimod cohorts as compared to placebo. The KPS and Vitality (SF-36 subscale) quality of life secondary endpoints demonstrated similar clinically significant improvements for the rintatolimod cohort as a function of the same ET dichotomization. Rintatolimod was generally well-tolerated in this CFS/ME population. Conclusions: Using a modified Bruce ET protocol with reduced physical exertion allowed clear identification of patient responders to rintatolimod with severe CFS/ME syndrome. Rintatolimod produced significant enhancement in ET and quality of life indicators in patients able to complete >9 minutes in a modified Bruce ET test. Rintatolimod also reduced deterioration in ET compared to placebo in patients with the poorest initial ET. Exercise endurance >9 minutes in a Bruce protocol modified for patients with CFS/ME provides a method to identify patients most likely to respond to rintatolimod.

The 18th International Conference on Antiviral Research (ICAR) was held at the Princess Sofia Hotel in Barcelona, Spain, from 11th – 14th April, 2005. This is a yearly international meeting sponsored by the International Society for Antiviral Research (ISAR). The current president of ISAR is John A Secrest 3rd of the Southern Research Institute. The scientific programme committee was chaired by John C Drach from the University of Michigan. ISAR was founded in 1987 to exchange prepublication basic, applied and clinical information on the development of antiviral, chemical and biological agents as well as to promote collaborative research. The ISAR has had a major role in the significant advances of the past decade in the reduction of the societal burdens of viral diseases by the focus of ICAR on the discovery and clinical application of antiviral agents. The 18th ICAR was organised as a series of focus presentations on specific viral groups consisting of oral and poster presentations of original research f...

A potentially contributing factor to the development and chronicity of pyoderma gangrenosum is infection with the relatively recently characterized human pathogen, Chlamydia pneumoniae. C pneumoniae is an obligate intracellular bacterium that can infect endothelial, monocyte, and smooth muscle cells and is associated with cardiopulmonary diseases. A case of serologically, polymerase chain reaction-positive, immunohistochemically, and culture-documented viable C pneumoniae organisms in a chronic pyoderma gangrenosum ulcer is reported, a finding that has not been described previously.

Children with learning difficulties have an increased prevalence of psychosocial adjustment problems. We hypothesized that within the context of a risk and resilience model contextual variables influence adjustment, over and above any effects of academic performance. The Behavior Assessment System for Children (BASC) was used to evaluate the relationship between psychosocial adjustment, changes in academic skills, and contextual factors longitudinally over a two–year period in 100 children (ages 7 to 11) referred for learning problems. Although there was little improvement in academic skills, contextual factors contributed to observed psychosocial outcome. Different factors were salient for parents, teachers, and children. Context–derived vulnerability and protective factors can be important determinants of adjustment in children with learning problems, even though the academic difficulties can remain chronic.

e14524 Background: Humoral tumor markers are used clinically for real-time assessment of therapeutic efficacy. In pancreatic ductal adenocarcinoma (PDAC) the predominant marker is CA19-9, which is not expressed by 10 to 30% of patients depending on race. We compared plasma cell-free DNA (cfDNA) copy number based assay with changes in serum CA19-9 levels and radiological responses to predict responses to systemic therapy. Methods: In a laboratory blinded, prospective multicenter pilot study, 40 non-resectable PDAC patients, treated with (m)FOLFIRINOX, CAPIRI, or gemcitabine +/- nab-paclitaxel) are currently enrolled. CA19-9 was determined in the local center’s laboratory. Tumor cfDNA was measured with a copy-number instability (CNI) scoring assay, determined by next generation sequencing in a centralized laboratory. The CNI score assesses the amount of cfDNA with somatic macro-alterations originating from malignant neoplasms. The difference of the values before commencing therapy (baseline) and prior to cycle 2 (either rising or falling) was calculated as a predictor of standardized radiological evaluation of chemotherapeutic efficacy. Results: 37 patients (3 drop-outs) had data for baseline and cycle 2, of which CA19-9 was elevated and evaluable in 29 patients. The direction from baseline to cycle 2 of CA19-9 and CNI scores were in agreement in 18/29 patients. 9 of 11 cases with discordant CNI score and CA19-9 had treatment response data, and CNI correlated with 7/9 (78%); in contrast 7/9 had rising CA19-9, when response was stable disease or better (22% concordance). In the 27 patients with available imaging, CNI predicted better (n = 18) than CA19-9 (n = 10) (p = 0.03 Fisher’s exact). Conclusions: This comparative study on cfDNA versus CA19-9 suggest that cfDNA CNI quantitation is a potentially more reliable blood based marker for early real-time assessment of efficacy in systemic PDAC therapy than CA19-9, compared to standard of care imaging. The better prediction after the first cycle might be due to the very short in vivo half-life of cfDNA ( < 1hr) compared to about one week for CA19-9. These results justify a larger prospective validation trial.

6055 Background: Copy number instability (CNI) signatures of cancers can be readily detected by Next Generation Sequencing of plasma cell-free DNA (cfDNA). HPV detected in oropharyngeal carcinomas is currently the only prognostic biomarker available. We report here CNI scores for disease monitoring of Head and Neck Cancers (HNC) with potential predictive value for personalized therapeutic options. Methods: A total of 132 plasma samples were collected from 54 HNC patients under informed consent and IRB approval. cfDNA was extracted from plasma, ~20M paired-end NGS mappable reads (reference: HG19) per sample were generated and CNI scores were calculated by read counting statistics. After unblinding CNI scores were evaluated as diagnostic parameter for association with disease characteristics and progression. Survival analysis was conducted after dichotomization of baseline CNI scores at a value of 31 corresponding to the 97.5thpercentile of a normal reference group (RG, n = 141). Results: CNI scores above the 97.5th RG percentile were detected in 40 out of 54 (74%) treatment naïve baseline samples. 29 patients with tumors ≤ T3 (62%, n = 42) and 11 out of 12 (92%) with T4 tumors had CNI scores > 31, with significantly higher CNI scores (p = 0.04) seen for T4 tumors. Higher CNI scores were also found in patients with tumor lymph node invasion (n = 37; median: 381, Q25-Q75: 57-1573) compared to negative lymph nodes (pN0, n = 17; 27, 19-64, p= 0.0004). A steep decline of CNI scores was detected after surgical resection, with increasing CNI scores in later disease progression. The pre-operative CNI scores were a stronger predictor of time to recurrence (p = 7*10-5) than the pN status (p = 0.05) (Cox regression). High baseline CNIs ( > 31) strongly correlated with time to recurrence (Kaplan-Meier log-rank p = 0.018) with a median of 20 months and median overall survival of 30 months in the high CNI group, neither reached in the low CNI-score group (60 m follow-up). Conclusions: Chromosomal instability within HNC was quantified from plasma cfDNA as CNI score. The CNI score may serve as better predictor for the time to recurrence interval than pN status. The data suggests that cfDNA analysis as CNI score may serve as real-time marker of treatment efficacy and outcome.

BACKGROUNDGenomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate.METHODSDNA was extracted from serum of 204 patients with prostate cancer (Gleason score 2–10), 207 male controls, and patients with benign hyperplasia (n = 10) and prostatitis (n = 10). DNA was amplified by use of random primers, tagged with molecular identifiers, sequenced on a SOLID system, and aligned to the human genome. We evaluated the number of sequence reads of cfDNA in sliding 100-kbp intervals for variation from controls. We used chromosomal regions with significant variations in alignment hits for their ability to segregate patients and matched controls.RESULTSUsing ROC curves to assess diagnostic performance, we evaluated the number of regions in a first subset (n = 177), with variations in alignment hits alone, provided an area under the curve (AUC) of 0.81 (95% CI 0.7–0.9, P < 0.001). Using 5 rounds of 10-fold cross-validation with the full data set, we established a final model that discriminated prostate cancer from controls with an AUC of 0.92 (0.87–0.95), reaching a diagnostic accuracy of 83%. Both benign prostatic hypertrophy and prostatitis could be distinguished from prostate cancer by use of cfDNA, with an accuracy of 90%.CONCLUSIONSAssessment of a limited number of chromosomal structural instabilities by use of massive parallel sequencing of cfDNA was sufficient to distinguish between prostate cancer and controls. This large cohort demonstrates the utility of cfDNA in prostate cancer recently established in other malignant neoplasms.

The genus, Chlamydophilia, as obligate intracellular pathogens, induce chronic scarring in humans. Chlamydia pneumoniae, a common cause of pneumonia, infects endothelial cells and circulating macrophages. Evidence that C. pneumoniae is an opportunistic pathogen in chronic skin ulcers and other inflammatory skin conditions analogous to its role in atherosclerosis is reviewed.

The novel avian H7N9 influenza virus has caused more than 130 human infections with 43 deaths (as of September, 2013) in China. Because of the lack of existing immunity against H7 subtype influenza viruses in the human population and the absence of a licensed commercial vaccine, antiviral drugs are critical tools for the treatment of infection with this novel H7N9. Both M2-ion channel blockers and neuraminidase inhibitors are used as antiviral drugs for influenza infections of humans. The emerging H7N9 viruses are resistant to the M2-ion channel blockers because of a S31N mutation in the M2 protein; additionally, some H7N9 isolates have gained neuraminidase R292K substitution resulting in broad resistance to neuraminidase inhibitors. In this study we report that Alferon N can inhibit wild type and 292K H7N9 viruses replication in vitro. Since Alferon N is approved for clinical use, this would allow a rapid regulatory approval process for this drug under pandemic threat.

The Diagnostic Rating Scale (DRS) was completed by the parents and teachers of 82 children referred for clinical evaluations, 73 referred children seen twice, and 218 non-referred children from the community. The DRS, which uses a categorical rather than a dimensional rating approach, was 70% to 90% sensitive to diagnoses of Attention Deficit/Hyperactivity Disorder (ADHD) made by blind clinical teams. In research and clinical applications, the DRS could improve screening efficiency, especially in situations where it would be desirable to exclude all children who might have ADHD or identify all children with Hyperactive-Impulsive symptoms. Because of its objectivity and consistency with the Diagnostic and Statistical Manual (DSM)-IV criteria, the DRS could facilitate comparison of participant samples across studies.

Our identification of Chlamydia pneumoniae in the cerebrospinal fluid (CSF) of a patient with multiple sclerosis (MS) led us to examine the incidence of this organism in the CSF from 17 patients with relapsing-remitting MS, 20 patients with progressive MS, and 27 patients with other neurological diseases (OND). CSF samples were examined for C pneumoniae by culture, polymerase chain reaction assays, and CSF immunoglobulin (Ig) reactivity with C pneumoniae elementary body antigens. C pneumoniae was isolated from CSF in 64% of MS patients versus 11% of OND controls. Polymerase chain reaction assays demonstrated the presence of C pneumoniae MOMP gene in the CSF of 97% of MS patients versus 18% of OND controls. Finally, 86% of MS patients had increased CSF antibodies to C pneumoniae elementary body antigens as shown by enzyme-linked immunosorbent assay absorbance values that were 3 SD greater than those seen in OND controls. The specificity of this antibody response was confirmed by western blot assays of the CSF, using elementary body antigens. Moreover, CSF isoelectric focusing followed by western blot assays revealed cationic antibodies against C pneumoniae. Infection of the central nervous system with C pneumoniae is a frequent occurrence in MS patients. Although the organism could represent the pathogenetic agent of MS, it may simply represent a secondary infection of damaged central nervous system tissue. A therapeutic trial directed at eliminating C pneumoniae from the central nervous system may provide additional information on its role in MS.

Background: The primary antigenic domain responsible for complement-mediated antibody-dependent enhancement (C′-ADE) of HIV and simian immunodeficiency virus resides in the principal immunodominant sequence of the transmembrane protein. Objective: To identify whether there are amino-acid residues common to the epitopes of the known enhancing human monoclonal antibodies (MAb), and to provide a structural model for this functional region present on the HIV envelope. Since our model predicts that this region is involved in the association of gp120 with gp41, this association was monitored for each mutant. Design: The binding of enhancing human MAb to point and deletion mutations within the enhancing domain was analyzed by two methods. The first analyzed binding to mutants expressed in COS cells: the second quantified the binding of four enhancing human MAb to each mutant gp160 versus wild-type control by enzyme-linked immunosorbent assay (ELISA). Methods: Site-directed mutagenesis was used to produce specific deletions and point mutants, which were expressed in COS cells. Binding of MAb 50–69 and V3-loop MAb 5F7 were visualized in the wild-type and each of the mutant constructs by immunohistochemistry. Quantitative evaluation of enhancing human MAb binding to each mutant versus wild-type was performed by ELISA. A model for the enhancing domain and its relationship to gp120 association with gp41 was provided by molecular dynamics and ligand docking methods. Results: All available enhancing human MAb known to bind to the principal immunodominant region of gp41 were unable to bind to deletions involving the disulfide loop, which in our molecular model provided the primary association site between gp120 and gp41. Point mutations in the loop blocked this association, but had a quantitatively smaller effect on the binding of the enhancing human MAb. A conservative W596Y mutation completely blocked the binding of all human MAb, but had no effect on gp120–gp41 association. Conclusions: A variety of mutations within the primary C′-ADE domain inhibit binding of enhancing human MAb as well as blocking the association of gp120 and gp41. A conservative W596Y mutation blocks binding of all enhancing human MAb with retention of gp120–gp41 association. These data are important to the design of vaccines in which the primary enhancing epitope is disarmed to prevent the subsequent induction of an amnestic response that could lead to viral enhancement of infection. The retention of the gp120–gp41 association is postulated to yield an immunogen similar to natural infection for both subunit and genetic vaccines.

Fifteen patients who fit the CDC definition of chronic fatigue syndrome (CFS) and had evidence of severe reduction in performance levels by low Kamofsky performance scores (KPS) of 20-60 were treated with Ampligen. At baseline most patients showed evidence of cerebral dysfunction by neuropsychological testing, were antigen positive by cell culture assay for human herpesvirus-6 (HHV-6), and displayed reduced performance during exercise tolerance testing, as measured by oxygen consumption. These patients represented a subset of CFS patients with especially severe and sustained symptomatology. Following 1248 weeks of Ampligen therapy, sustained improvements were noted in KPS (p < 0.01). Cognitive function improved including IQ and memory. Oxygen uptake and treadmill duration during exercise tolerance testing was also improved after 24 weeks of treatment (p < 0.01). Reduction in HHV-6 expression as measured by the giant cell assay was significant (p < 0.001). Patients continued to show significant improvement...

Four of eight human monoclonal antibodies (huMAbs) to gp41 were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp41 transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp41 which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection.

A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

A preliminary study is made of simultaneous and lagged seasonal cross correlations between northern Australian sea level and district rainfall anomalies. Zero-lagged patterns show strong links with the El Nino/Southern Oscillation (ENSO) phenomenon over northern and eastern Australia during austral winter-spring seasons. These findings are similar to, but more robust than, those reported for simultaneous cross correlations between Australian district rainfall and either the southern oscillation index, Darwin mean sea level pressure, or northern Australian sea surface temperatures. This appears to be a consequence of the strong integrating response of northern Australian sea levels, particularly to inverse barometric and wind forcings on seasonal time scales. Such relationships are explored further in lagged cross correlations. As observed in other studies of seasonal persistence associated with ENSO and anti-ENSO phases, rainfall precursors extend in time from one to two seasons. However. signifi...

The antiseptics 4% chlorhexidine gluconate detergent formulation containing 4% isopropyl alcohol (Hibiclens/Hibiscrub) and 0.5% chlorhexidine gluconate in 70% isopropyl alcohol with emollients (Hibistat/Hibisol) efficiently inactivated human immunodeficiency virus (HIV) produced in cell culture within 15 seconds. These antiseptics were completely effective at 1:100 and 1:5 dilutions, respectively. Therefore, use of these products according to the manufacturer's instructions (i.e. undiluted) for routine disinfection of hands following contact with HIV-contaminated materials, as well as immediate disinfection of abrasions or cuts exposed to HIV, should have significant protective effects.