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1.  Aggregate complexes of HIV-1 induced by multimeric antibodies 
Retrovirology  2014;11(1):78.
Background
Antibody mediated viral aggregation may impede viral transfer across mucosal surfaces by hindering viral movement in mucus, preventing transcytosis, or reducing inter-cellular penetration of epithelia thereby limiting access to susceptible mucosal CD4 T cells and dendritic cells. These functions may work together to provide effective immune exclusion of virus from mucosal tissue; however little is known about the antibody characteristics required to induce HIV aggregation. Such knowledge may be critical to the design of successful immunization strategies to facilitate viral immune exclusion at the mucosal portals of entry.
Results
The potential of neutralizing and non-neutralizing IgG and IgA monoclonals (mAbs) to induce HIV-1 aggregation was assessed by Dynamic light scattering (DLS). Although neutralizing and non-neutralizing IgG mAbs and polyclonal HIV-Ig efficiently aggregated soluble Env trimers, they were not capable of forming viral aggregates. In contrast, dimeric (but not monomeric) IgA mAbs induced stable viral aggregate populations that could be separated from uncomplexed virions. Epitope specificity influenced both the degree of aggregation and formation of higher order complexes by dIgA. IgA purified from serum of uninfected RV144 vaccine trial responders were able to efficiently opsonize viral particles in the absence of significant aggregation, reflective of monomeric IgA.
Conclusions
These results collectively demonstrate that dIgA is capable of forming stable viral aggregates providing a plausible basis for testing the effectiveness of aggregation as a potential protection mechanism at the mucosal portals of viral entry.
Electronic supplementary material
The online version of this article (doi:10.1186/s12977-014-0078-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12977-014-0078-8
PMCID: PMC4193994  PMID: 25274446
HIV-1; Mucosal immunity; Immunoglobulin A; Aggregation
2.  A computational framework for the analysis of peptide microarray antibody binding data with application to HIV vaccine profiling 
We present an integrated analytical method for analyzing peptide microarray antibody binding data, from normalization through subject-specific positivity calls and data integration and visualization. Current techniques for the normalization of such data sets do not account for non-specific binding activity. A novel normalization technique based on peptide sequence information quickly and effectively reduced systematic biases. We also employed a sliding mean window technique that borrows strength from peptides sharing similar sequences, resulting in reduced signal variability. A smoothed signal aided in the detection of weak antibody binding hotspots. A new principled FDR method of setting positivity thresholds struck a balance between sensitivity and specificity. In addition, we demonstrate the utility and importance of using baseline control measurements when making subject-specific positivity calls. Data sets from two human clinical trials of candidate HIV-1 vaccines were used to validate the effectiveness of our overall computational framework.
doi:10.1016/j.jim.2013.06.001
PMCID: PMC3999921  PMID: 23770318
Peptide microarrays; Antibodies; Normalization; Positivity calls; Software; Visualization
3.  Activation of the Wnt pathway through Wnt2 promotes metastasis in pancreatic cancer 
Wnt signaling pathway plays an important role in physiological and pathological process, including in the occurrence and development of tumor. The purpose of this study is to determine whether Wnt2 and sFRP4, key molecules of signaling pathway, are of prognostic value for survival in patients with pancreatic cancer. We performed immunohistochemistry on tissue microarrays containing 90 pancreatic cancer specimens to evaluate the protein expression of Wnt2 and sFRP4. Our results showed that the cytoplasmic expression level of Wnt2 in pancreatic cancer tissues was significantly associated with LNM (P=0.029) and AJCC stage (P=0.008). Additionally, Kaplan-Meier analysis indicated that high Wnt2 expression was significantly correlated with poor clinical outcomes of patients with pancreatic cancer. In conclusion, Wnt2 may play an important role in the development of pancreatic cancer through activation of the Wnt pathways and serve as a potential candidate for treatment target of pancreatic cancer.
PMCID: PMC4163618  PMID: 25232495
Pancreatic cancer; Wnt signalling pathway; Wnt2; sFRP4; immunohistochemistry; survival analysis
5.  Cytoplasmic HSP90α expression is associated with perineural invasion in pancreatic cancer 
Pancreatic cancer (PC) is an aggressive and devastating disease with a dismal prognosis. The study aimed to investigate the role of HSP90α and PDIA3 in patients with PC. Immunohistochemistry was performed on tissue microarrays containing 186 pairs of PC and normal pancreatic tissues to assess the expression levels of HSP90α and PDIA3. The expression levels of cytoplasmic HSP90α (P = 0.032) and PDIA3 (P = 0.043) in PCs were significantly higher than those in normal pancreas tissues, but nuclear HSP90α showed lower expression in PC tissues (P = 0.002). In addition, cytoplasmic expression of HSP90α and PDIA3 was significantly associated with perineural invasion (PNI) (P = 0.004) and sex (P = 0.014), respectively. These results indicate that cytoplasmic HSP90α may serve as a biomarker for PNI in PCs.
PMCID: PMC4097286  PMID: 25031753
Pancreatic cancer; HSP90α; PDIA3; perineural invasion
6.  Capacity for Infectious HIV-1 Virion Capture Differs by Envelope Antibody Specificity 
Journal of Virology  2014;88(9):5165-5170.
Antibody capacity to recognize infectious virus is a prerequisite of many antiviral functions. We determined the infectious virion capture index (IVCI) of different antibody specificities. Whereas broadly neutralizing antibodies (bNAbs), except for an MPER bNAb, selectively captured infectious virions, non-bNAbs and mucosal human immunodeficiency virus type 1 (HIV-1)-positive IgG captured subsets of both infectious and noninfectious virions. Infectious virion capture was additive with a mixture of antibodies, providing proof of concept for vaccine-induced antibodies that together have improved capacity to recognize infectious virions.
doi:10.1128/JVI.03765-13
PMCID: PMC3993833  PMID: 24554654
7.  Perspectives from Nurse Managers on Informatics Competencies 
The Scientific World Journal  2014;2014:391714.
Background and Purpose. Nurse managers are in an excellent position for providing leadership and support within the institutions they serve and are often responsible for accessing information that is vital to the improvement of health facility processes and patients' outcomes. Therefore, competency in informatics is essential. The purposes of this study are to examine current informatics competency levels of nurse managers and to identify the variables that influence these competencies. Methods. A questionnaire designed to assess demographic information and nursing informatics competency was completed by 68 nurse managers. Multiple linear regression analysis was conducted to analyze the factors influencing informatics competency. Results. Descriptive analysis of the data revealed that informatics competency of these nurse managers was in the moderate range (77.65 ± 8.14). Multiple linear regression analysis indicated that level of education, nursing administration experience, and informatics education/training were significant factors affecting competency levels. Conclusion. The factors identified in this study can serve as a reference for nurse managers who were wishing to improve their informatics competency, hospital administrators seeking to provide appropriate training, and nursing educators who were making decisions about nursing informatics curricula. These findings suggest that efforts to enhance the informatics competency of nurse managers have marked potential benefits.
doi:10.1155/2014/391714
PMCID: PMC3982287  PMID: 24790565
8.  DNA and Protein Co-Immunization Improves the Magnitude and Longevity of Humoral Immune Responses in Macaques 
PLoS ONE  2014;9(3):e91550.
We tested the concept of combining DNA with protein to improve anti-HIV Env systemic and mucosal humoral immune responses. Rhesus macaques were vaccinated with DNA, DNA&protein co-immunization or DNA prime followed by protein boost, and the magnitude and mucosal dissemination of the antibody responses were monitored in both plasma and mucosal secretions. We achieved induction of robust humoral responses by optimized DNA vaccination delivered by in vivo electroporation. These responses were greatly increased upon administration of a protein boost. Importantly, a co-immunization regimen of DNA&protein injected in the same muscle at the same time induced the highest systemic binding and neutralizing antibodies to homologous or heterologous Env as well as the highest Env-specific IgG in saliva. Inclusion of protein in the vaccine resulted in more immunized animals with Env-specific IgG in rectal fluids. Inclusion of DNA in the vaccine significantly increased the longevity of systemic humoral immune responses, whereas protein immunization, either as the only vaccine component or as boost after DNA prime, was followed by a great decline of humoral immune responses overtime. We conclude that DNA&protein co-delivery in a simple vaccine regimen combines the strength of each vaccine component, resulting in improved magnitude, extended longevity and increased mucosal dissemination of the induced antibodies in immunized rhesus macaques.
doi:10.1371/journal.pone.0091550
PMCID: PMC3953433  PMID: 24626482
9.  Expression and prognostic value of c-Myc and Fas (CD95/APO1) in patients with pancreatic cancer 
The identification of molecular prognostic markers for pancreatic cancer patients could provide insightful information for their management in the clinic. The aim of the study is to investigate whether or not the expressions of c-Myc and Fas (CD95/APO1) have prognostic relevance for overall survival (OS) in patients with pancreatic cancer. we used immunohistochemistry on tissue microarrays containing 162 pancreatic cancer specimens to assess the protein expression levels of c-Myc and Fas. Kaplan-Meier survival analysis demonstrated that high level of c-Myc cytoplasmic expression was significantly correlated with worse survival in patients with pancreatic cancer (P = 0.012), while high level of Fas cytoplasmic expression was significantly associated with better outcome of pancreatic cancer (P = 0.046). However, multivariate Cox model analysis showed that tumor differentiation, lymph node status and c-Myc cytoplasmic expression were significant independent prognostic factors for OS (P < 0.001, P = 0.023, P = 0.001, respectively). On the contrary, Fas cytoplasmic expression did not independently influence patient’s prognosis (P = 0.249). Our data suggested that high level of c-Myc cytoplasmic expression may be considered as a valuable marker for prognosis of pancreatic cancer.
PMCID: PMC3925922  PMID: 24551298
Pancreatic cancer; c-Myc; Fas (CD95/APO1); immunohistochemistry; survival analysis
10.  The Thai Phase III HIV Type 1 Vaccine Trial (RV144) Regimen Induces Antibodies That Target Conserved Regions Within the V2 Loop of gp120 
AIDS Research and Human Retroviruses  2012;28(11):1444-1457.
Abstract
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
doi:10.1089/aid.2012.0103
PMCID: PMC3484815  PMID: 23035746
11.  Plasma IgG to Linear Epitopes in the V2 and V3 Regions of HIV-1 gp120 Correlate with a Reduced Risk of Infection in the RV144 Vaccine Efficacy Trial 
PLoS ONE  2013;8(9):e75665.
Neutralizing and non-neutralizing antibodies to linear epitopes on HIV-1 envelope glycoproteins have potential to mediate antiviral effector functions that could be beneficial to vaccine-induced protection. Here, plasma IgG responses were assessed in three HIV-1 gp120 vaccine efficacy trials (RV144, Vax003, Vax004) and in HIV-1-infected individuals by using arrays of overlapping peptides spanning the entire consensus gp160 of all major genetic subtypes and circulating recombinant forms (CRFs) of the virus. In RV144, where 31.2% efficacy against HIV-1 infection was seen, dominant responses targeted the C1, V2, V3 and C5 regions of gp120. An analysis of RV144 case-control samples showed that IgG to V2 CRF01_AE significantly inversely correlated with infection risk (OR= 0.54, p=0.0042), as did the response to other V2 subtypes (OR=0.60-0.63, p=0.016-0.025). The response to V3 CRF01_AE also inversely correlated with infection risk but only in vaccine recipients who had lower levels of other antibodies, especially Env-specific plasma IgA (OR=0.49, p=0.007) and neutralizing antibodies (OR=0.5, p=0.008). Responses to C1 and C5 showed no significant correlation with infection risk. In Vax003 and Vax004, where no significant protection was seen, serum IgG responses targeted the same epitopes as in RV144 with the exception of an additional C1 reactivity in Vax003 and infrequent V2 reactivity in Vax004. In HIV-1 infected subjects, dominant responses targeted the V3 and C5 regions of gp120, as well as the immunodominant domain, heptad repeat 1 (HR-1) and membrane proximal external region (MPER) of gp41. These results highlight the presence of several dominant linear B cell epitopes on the HIV-1 envelope glycoproteins. They also generate the hypothesis that IgG to linear epitopes in the V2 and V3 regions of gp120 are part of a complex interplay of immune responses that contributed to protection in RV144.
doi:10.1371/journal.pone.0075665
PMCID: PMC3784573  PMID: 24086607
12.  Infectious Virion Capture by HIV-1 gp120-Specific IgG from RV144 Vaccinees 
Journal of Virology  2013;87(14):7828-7836.
The detailed examination of the antibody repertoire from RV144 provides a unique template for understanding potentially protective antibody functions. Some potential immune correlates of protection were untested in the correlates analyses due to inherent assay limitations, as well as the need to keep the correlates analysis focused on a limited number of endpoints to achieve statistical power. In an RV144 pilot study, we determined that RV144 vaccination elicited antibodies that could bind infectious virions (including the vaccine strains HIV-1 CM244 and HIV-1 MN and an HIV-1 strain expressing transmitted/founder Env, B.WITO.c). Among vaccinees with the highest IgG binding antibody profile, the majority (78%) captured the infectious vaccine strain virus (CM244), while a smaller proportion of vaccinees (26%) captured HIV-1 transmitted/founder Env virus. We demonstrated that vaccine-elicited HIV-1 gp120 antibodies of multiple specificities (V3, V2, conformational C1, and gp120 conformational) mediated capture of infectious virions. Although capture of infectious HIV-1 correlated with other humoral immune responses, the extent of variation between these humoral responses and virion capture indicates that virion capture antibodies occupy unique immunological space.
doi:10.1128/JVI.02737-12
PMCID: PMC3700223  PMID: 23658446
13.  Vaccination with Vaxfectin® adjuvanted SIV DNA induces long-lasting humoral immune responses able to reduce SIVmac251 Viremia 
Human Vaccines & Immunotherapeutics  2013;9(10):2069-2080.
We evaluated the immunogenicity and efficacy of Vaxfectin® adjuvanted SIV DNA vaccines in mice and macaques. Vaccination of mice with Vaxfectin® adjuvanted SIV gag DNA induced higher humoral immune responses than administration of unadjuvanted DNA, whereas similar levels of cellular immunity were elicited. Vaxfectin® adjuvanted SIVmac251 gag and env DNA immunization of rhesus macaques was used to examine magnitude, durability, and efficacy of humoral immunity. Vaccinated macaques elicited potent neutralizing antibodies able to cross-neutralize the heterologous SIVsmE660 Env. We found remarkable durability of Gag and Env humoral responses, sustained during ~2 y of follow-up. The Env-specific antibody responses induced by Vaxfectin® adjuvanted env DNA vaccination disseminated into mucosal tissues, as demonstrated by their presence in saliva, including responses to the V1-V2 region, and rectal fluids. The efficacy of the immune responses was evaluated upon intrarectal challenge with low repeated dose SIVmac251. Although 2 of the 3 vaccinees became infected, these animals showed significantly lower peak virus loads and lower chronic viremia than non-immunized infected controls. Thus, Vaxfectin® adjuvanted DNA is a promising vaccine approach for inducing potent immune responses able to control the highly pathogenic SIVmac251.
doi:10.4161/hv.25442
PMCID: PMC3906391  PMID: 23820294
HIV; Rhesus macaques; SIVmac239; SIVsmE660; V1 and V2 antibodies; adjuvant; antibody; avidity; mucosal immunity; neutralizing antibody; rectal fluid; saliva; systemic immunity
14.  UNC51-like kinase 1 as a potential prognostic biomarker for hepatocellular carcinoma 
Autophagy is a fundamental cell biological process that confers stress tolerance, limits damage, and sustains viability under adverse conditions. Defective autophagy is associated with diverse diseases. The study aimed to investigate the relationship between UNC51-like kinase 1 (ULK1) expression and clinicopathological characteristics as well as survival in patients with hepatocellular carcinoma (HCC). Expression levels of ULK1 in 55 paired HCC and paracancerous tissues were examined using immunohistochemistry. Although not statistically significant, the expression of ULK1 in adjacent peritumoural tissue was lower than those in HCC tissues (P = 0.113). Expression level of ULK1 was significantly associated with tumor size (P = 0.015) after adjusted for age, sex, histologic grade, cirrhosis and TNM. Survival analysis showed that patients with high ULK1 expression had worse survival time than those with low ULK1 expression (hazard rate = 2.684, 95% CI 1.029–7.006, P = 0.044). The findings of the present study provide evidence that ULK1 represents a potential novel prognostic biomarker for HCC patients and may play an important role during the progression of HCC.
PMCID: PMC3606861  PMID: 23573318
Hepatocellular carcinoma; autophagy; UNC51-like kinase 1; survival; biomarker
15.  Analysis of V2 Antibody Responses Induced in Vaccinees in the ALVAC/AIDSVAX HIV-1 Vaccine Efficacy Trial 
PLoS ONE  2013;8(1):e53629.
The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.
doi:10.1371/journal.pone.0053629
PMCID: PMC3547933  PMID: 23349725
16.  Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial 
The New England Journal of Medicine  2012;366(14):1275-1286.
BACKGROUND
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
METHODS
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
RESULTS
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
CONCLUSIONS
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
doi:10.1056/NEJMoa1113425
PMCID: PMC3371689  PMID: 22475592
17.  RhoT1 and Smad4 Are Correlated with Lymph Node Metastasis and Overall Survival in Pancreatic Cancer 
PLoS ONE  2012;7(7):e42234.
Cancer cell invasion and metastasis are the most important adverse prognostic factors for pancreatic cancer. Identification of biomarkers associated with outcome of pancreatic cancer may provide new approaches and targets for anticancer therapy. The aim of this study is to examine the relationship between the expression of RhoT1, Smad4 and p16 and metastasis and survival in patients with pancreatic cancer. The analysis showed that the high cytoplasmic expression levels of RhoT1, Smad4 and p16 in pancreatic cancer tissues had significantly negative correlation with lymph node metastasis (LNM) (P = 0.017, P = 0.032, P = 0.042, respectively). However, no significant association was observed between perineural invasion (PNI) and the expression of above three proteins (all P>0.05). Additionally, the survival analysis showed that the low expression levels of RhoT1 and Smad4 were significantly associated with worse survival (P = 0.034, P = 0.047, respectively). In conclusion, these results indicated that the low-expression levels of RhoT1 and Smad4 were significantly associated with LNM and shorter survival. RhoT1 may be considered as a potential novel marker for predicting the outcome in patients with pancreatic cancer.
doi:10.1371/journal.pone.0042234
PMCID: PMC3409151  PMID: 22860091
18.  Analysis of whole genomic expression profiles and screening of the key signaling pathways associated with pancreatic cancer 
The tumorigenesis of pancreatic cancer is thought to be a complex process. Investigation of the molecular mechanism of pancreatic cancer and exploring the specific markers for early diagnosis and specific targets of therapy is a key point to prevent and treat pancreatic cancer effectively and to improve their prognosis. In this study, expression profiles experiment was performed using Agilent human whole genomic oligonucleotide microarrays with 41,000 genes. Differentially expressed genes related with pancreatic cancer were screened, and analyzed further by GO term analysis and KEGG Pathway analysis. Our results showed that there were 1276 differentially expressed genes associated with pancreatic cancer. 691 genes were up regulated and 585 were down regulated in pancreatic cancer group. The present study confirmed that the occurrence of pancreatic cancer was involved in multiple-gene interaction. In addition, our study found that pancreatic cancer was related to an activation of the mTOR signaling pathway and renal cell carcinoma pathway.
PMCID: PMC3430114  PMID: 22949936
Pancreatic cancer; microarray; gene expression profile; signaling pathway
19.  Isolation of HIV-1-Neutralizing Mucosal Monoclonal Antibodies from Human Colostrum 
PLoS ONE  2012;7(5):e37648.
Background
Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses.
Methods
We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs).
Results
The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization.
Conclusions
These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.
doi:10.1371/journal.pone.0037648
PMCID: PMC3356285  PMID: 22624058
20.  Polyclonal B Cell Responses to Conserved Neutralization Epitopes in a Subset of HIV-1-Infected Individuals▿† 
Journal of Virology  2011;85(21):11502-11519.
A small proportion of HIV-infected individuals generate a neutralizing antibody (NAb) response of exceptional magnitude and breadth. A detailed analysis of the critical epitopes targeted by broadly neutralizing antibodies should help to define optimal targets for vaccine design. HIV-1-infected subjects with potent cross-reactive serum neutralizing antibodies were identified by assaying sera from 308 subjects against a multiclade panel of 12 “tier 2” viruses (4 each of subtypes A, B, and C). Various neutralizing epitope specificities were determined for the top 9 neutralizers, including clade A-, clade B-, clade C-, and clade A/C-infected donors, by using a comprehensive set of assays. In some subjects, neutralization breadth was mediated by two or more antibody specificities. Although antibodies to the gp41 membrane-proximal external region (MPER) were identified in some subjects, the subjects with the greatest neutralization breadth targeted gp120 epitopes, including the CD4 binding site, a glycan-containing quaternary epitope formed by the V2 and V3 loops, or an outer domain epitope containing a glycan at residue N332. The broadly reactive HIV-1 neutralization observed in some subjects is mediated by antibodies targeting several conserved regions on the HIV-1 envelope glycoprotein.
doi:10.1128/JVI.05363-11
PMCID: PMC3194956  PMID: 21849452
21.  Cross-Reactive HIV-1-Neutralizing Human Monoclonal Antibodies Identified from a Patient with 2F5-Like Antibodies ▿ †  
Journal of Virology  2011;85(21):11401-11408.
The genes encoding broadly HIV-1-neutralizing human monoclonal antibodies (MAbs) are highly divergent from their germ line counterparts. We have hypothesized that such high levels of somatic hypermutation could pose a challenge for elicitation of the broadly neutralizing (bn) Abs and that identification of less somatically mutated bn Abs may help in the design of effective vaccine immunogens. In a quest for such bn Abs, phage- and yeast-displayed antibody libraries, constructed using peripheral blood mononuclear cells (PBMCs) from a patient with bn serum containing Abs targeting the epitope of the bn MAb 2F5, were panned against peptides containing the 2F5 epitope and against the HIV-1 gp140JR-FL. Two MAbs (m66 and m66.6) were identified; the more mutated variant (m66.6) exhibited higher HIV-1-neutralizing activity than m66, although it was weaker than 2F5 in a TZM-bl cell assay. Binding of both MAbs to gp41 alanine substitution mutant peptides required the DKW664–666 core of the 2F5 epitope and two additional upstream residues (L660,663). The MAbs have long (21-residue) heavy-chain third complementarity-determining regions (CDR-H3s), and m66.6 (but not m66) exhibited polyspecific reactivity to self- and non-self-antigens. Both m66 and m66.6 are significantly less divergent from their germ line Ab counterparts than 2F5—they have a total of 11 and 18 amino acid changes, respectively, from the closest VH and Vκ germ line gene products compared to 25 for 2F5. These new MAbs could help explore the complex maturation pathways involved in broad neutralization and its relationship with auto- and polyreactivity and may aid design of vaccine immunogens and development of therapeutics against HIV-1 infection.
doi:10.1128/JVI.05312-11
PMCID: PMC3194990  PMID: 21880764
22.  The PIWI protein acts as a predictive marker for human gastric cancer 
Purpose
To investigate the expression of the human PIWI subfamily proteins in gastric cancer and their potential roles in the occurrence, development and prognosis of gastric cancer.
Methods and patients
Expression of the PIWI proteins were assessed by immunohistochemistry (IHC) in tissue microarrays (TMA), containing paired tumor tissue and adjacent non-cancer tissue from 182 patients who had undergone surgery in hospital for histologically proven gastric cancer (GC). Prognostic value and correlation with other clinicopathologic factors were evaluated in two classifications.
Results
The expression of PIWIL1-4 was significantly higher in tumor tissue than that in adjacent tissue; A significant correlation was observed between the higher expression of PIWI protein with the T stage, lymph node metastasis and clinical TNM (cTNM); Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that elevated PIWIL1 and PIWIL2 expression in cancer tissue predicted poorer overall survival (OS) compared with group in lower expression (36.5% VS 67.6%; 37.4% VS 54.2%; respectively). Notably, multivariate analyses by Cox’s proportional hazard model revealed that expression of PIWIL1 was an independent prognostic factor in gastric cancer.
Conclusions
The PIWI subfamily protein is an absolutely key molecular factor along with the tumor occurrence and development. And the PIWI protein could act as a potential biomarker for prognosis evaluation of gastric cancer.
PMCID: PMC3365820  PMID: 22670175
Argonaute protein; tissue microarray; Immunohistochemistry; gastric cancer
23.  Expression of c-Myc and Fas correlates with perineural invasion of pancreatic cancer 
Lymph node metastasis (LNM) and perineural invasion (PNI) are regarded as two important prognostic factors in pancreatic cancer. The aim of this study was to identify the LNM-associated and PNI-associated markers in pancreatic cancer. We have constructed a formalin-fixed paraffin embedded pancreatic tissue microarrays containing 324 cylindrical tissue cores of human pancreatic cancer and its paracancerous nonmalignant pancreatic specimens (NMPs) from 162 patients. Among those patients, there were 83 of 162 with PNI, and 65 of 162 with LNM. The protein products of 2 genes encompassing a variety of functional classes, such as oncogenes (c-Myc), apoptosis (Fas), were analyzed by immunohistochemistry on the tissue microarray. There was marked increase in cancer tissues cytoplasmic c-Myc expression level in pancreatic cancer compared with the NMPs (P < 0.0001). Additionally, the expression level of c-Myc was also significant increase in pancreatic cancer with PNI compared with cancer without PNI (P < 0.01). In contrast, cytoplasmic Fas, low expressed in pancreatic cancer (P < 0.0001) was negatively correlated with PNI (P < 0.05). Logistic regression analysis showed that the c-Myc expression in the cancer tissues cytoplasm acted as a potential and independently predictive factor for PNI. However, there were no significant association between the expression of these two genes and LNM (P > 0.05). This study for the first time described expression levels of c-Myc and Fas played an important role in PNI of pancreatic cancer. Combined detection can be used as predictive factors, especially c-Myc.
PMCID: PMC3365821  PMID: 22670178
Pancreatic cancer; perineural invasion; c-Myc; Fas; tissue microarrays
24.  ERCC1 C118T associates with response to FOLFOX4 chemotherapy in colorectal cancer patients in Han Chinese 
Background
Genetic variations influence treatment outcomes in cancer patients treated with chemotherapy. Detection of pharmacogenetic markers associated with treatment response may enable doctor to plan more precise and effective treatment tailoring to individual cancer patients.
Methods
A novel oligonucleotide microarray was developed to genotype 13 variations (DPYD*2, DPYD*5, DPYD*9, TYMS 6 bp Ins/Del, UGT1A1*6, UGT1A1*27, UGT1A1*28, GSTP1 Ile105Val, XRCC1 Arg399Gln, MTHFR C677T, MDR1 C3435T/A, MDR1 G2677A/T and ERCC1 C118T). The accuracy of genotypes obtained by microarray was assessed by independent sequencing. 73 patients first diagnosed with colorectal cancer (CRC) were treated with FOLFOX4 chemotherapy.
Results
All genotypes were successfully called by microarray, and were consistent with those identified by independent sequencing except two TYMS 6 bp Ins/Del genotypes. Patients with CT or TT genotype exhibited a higher probability of response to treatment than those with CC genotype. No other SNP was found to be associated with treatment response. Furthermore, these SNPs showed no associations with gastrointestinal, hematological or neurological toxicity.
Conclusions
ERCC1 C118T may be a predictive marker of treatment response to 5-FU/platinum chemotherapy for CRC. The microarray can significantly facilitate the process of detecting genetic variations and may help doctor plan more effective medication for individual cancer patient.
PMCID: PMC3342712  PMID: 22567180
Single nucleotide polymorphism; 5-FU; platinum; drug response; ERCC1; molecular biomarker
25.  HIV-Specific Functional Antibody Responses in Breast Milk Mirror Those in Plasma and Are Primarily Mediated by IgG Antibodies ▿ 
Journal of Virology  2011;85(18):9555-9567.
Despite months of mucosal virus exposure, the majority of breastfed infants born to HIV-infected mothers do not become infected, raising the possibility that immune factors in milk inhibit mucosal transmission of HIV. HIV Envelope (Env)-specific antibodies are present in the milk of HIV-infected mothers, but little is known about their virus-specific functions. In this study, HIV Env-specific antibody binding, autologous and heterologous virus neutralization, and antibody-dependent cell cytotoxicity (ADCC) responses were measured in the milk and plasma of 41 HIV-infected lactating women. Although IgA is the predominant antibody isotype in milk, HIV Env-specific IgG responses were higher in magnitude than HIV Env-specific IgA responses in milk. The concentrations of anti-HIV gp120 IgG in milk and plasma were directly correlated (r = 0.75; P < 0.0001), yet the response in milk was 2 logarithm units lower than in plasma. Similarly, heterologous virus neutralization (r = 0.39; P = 0.010) and ADCC activity (r = 0.64; P < 0.0001) in milk were directly correlated with that in the systemic compartment but were 2 log units lower in magnitude. Autologous neutralization was rarely detected in milk. Milk heterologous virus neutralization titers correlated with HIV gp120 Env-binding IgG responses but not with IgA responses (r = 0.71 and P < 0.0001, and r = 0.17 and P = 0.30). Moreover, IgGs purified from milk and plasma had equal neutralizing potencies against a tier 1 virus (r = 0.65; P < 0.0001), whereas only 1 out of 35 tested non-IgG milk fractions had detectable neutralization. These results suggest that plasma-derived IgG antibodies mediate the majority of the low-level HIV neutralization and ADCC activity in breast milk.
doi:10.1128/JVI.05174-11
PMCID: PMC3165739  PMID: 21734046

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