Plasma Nitric Oxide Consumption Is Elevated and Associated With Adverse Outcomes in Critically Ill Patients.

OBJECTIVES: Impaired nitric oxide (NO) bioavailability may contribute to microvascular dysfunction in sepsis. Excessive plasma NO consumption has been attributed to scavenging by circulating cell-free hemoglobin. This may be a mechanism for NO deficiency in sepsis and critical illness. We hypothesized that plasma NO consumption is high in critically ill patients, particularly those with sepsis, acute respiratory distress syndrome (ARDS), shock, and in hospital nonsurvivors. We further hypothesized that plasma NO consumption is correlated with plasma cell-free hemoglobin concentration. DESIGN: Retrospective cohort study. SETTING: Adult ICUs of an academic medical center. PATIENTS AND SUBJECTS: Three hundred sixty-two critically ill patients and 46 healthy control subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma NO consumption was measured using reductive chemiluminescence and cell-free hemoglobin was measured with a colorimetric assay. Mean (95% CI) plasma NO consumption (µM) was higher in critically ill patients versus healthy control subjects (3.9 [3.7-4.1] vs 2.1 [1.8-2.5]), septic versus nonseptic patients (4.1 [3.8-4.3] vs 3.6 [3.3-3.8]), ARDS versus non-ARDS patients (4.4 [4.0-4.9] vs 3.7 [3.6-3.9]), shock vs nonshock patients (4.4 [4.0-4.8] vs 3.6 [3.4-3.8]), and hospital nonsurvivors versus survivors (5.3 [4.4-6.4] vs 3.7 [3.6-3.9]). These relationships remained significant in multivariable analyses. Plasma cell-free hemoglobin was weakly correlated with plasma NO consumption. CONCLUSIONS: Plasma NO consumption is elevated in critically ill patients and independently associated with sepsis, ARDS, shock, and hospital death. These data suggest that excessive intravascular NO scavenging characterizes sepsis and adverse outcomes of critical illness.

Alteration of inhibitory and activating NK cell receptor expression on NK cells in HIV-infected Chinese.

Natural killer (NK) cell function, based on the expression of activating and inhibitory natural killer receptors (NKRs), may become abnormal during human immunodeficiency virus (HIV) infection. In this study, we investigated changes in receptor expression with individual and combinational analysis on NK cell subsets in HIV-infected Chinese. The results showed that natural killer group 2 member D (NKG2D) expression on total NK cells decreased significantly in HIV infection, while the expressions of natural killer group 2 member A (NKG2A) and killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail 1 (KIR3DL1) on total NK cells were not significantly different between any of the groups including HIV-positive treatment-naïve group, AIDS treatment-naïve group, HAART-treatment AIDS group and HIV-negative control group. Individual analysis of NKG2A(+) and KIR3DL1(+) cells revealed no significant differences in expression in any NK cell subsets between any of the groups, but the combinational analysis of NKG2D(-)NKG2A(+), and NKG2D(-)KIR3DL1(+) on the NK CD56(dim) cell subset in the AIDS group were increased compared to the HIV-negative control group. On the contrary, NKG2D(-)NKG2A(+) expression on the CD56(bright) subset decreased in the AIDS group compared to the control group. Highly active antiretroviral therapy (HAART) treatment almost completely restored the levels of these receptor expressions. The results indicate that the distinct alteration of activating and inhibitory NKR expression on NK cells and its subsets occurred during HIV progression. Moreover, the imbalanced change of activating and inhibitory NKRs on NK cells and its subsets may explain the impaired NK cell immunity in HIV infected individuals.

Alteration of inhibitory and activating natural killer cell receptor expression on T cells in human immunodeficiency virus-infected Chinese.

T cell expression of NKRs can trigger or inhibit cell-mediated cytotoxicity. However, few studies on T lymphocyte NKR expression in HIV infection exist. Here, we examined the expression patterns of NKG2D, NKG2A, and KIR3DL1 on CD8⁺ and CD3⁺CD8⁻ cells by multicolor flow cytometry in groups of patients with HIV, AIDS or HAART-treated AIDS, as well as HIV-negative normal controls. Individual analysis of KIR3DL1 on CD3⁺ CD8⁺ or CD3⁺CD8⁻ cells revealed no significant differences among any of the groups (P > 0.05). In contrast, the percentage of NKG2A⁺NKG2D⁻CD8⁺ T cells was higher in the AIDS group than in the HIV-negative normal control group (P < 0.01). Meanwhile, the prevalence of NKG2D⁺ NKG2A⁻ CD8⁺ T cells was lower in the AIDS group than in HIV-negative normal controls (P < 0.001). Similar results were also observed for the percentage of NKG2A⁺ NKG2D⁻ on CD3⁺ CD8⁻ cells. However, in contrast to CD8⁺ T cells, the frequencies of NKG2D⁺ NKG2A⁻ on CD3⁺CD8⁻ cells were higher in AIDS and HIV patients than in HIV-negative normal controls (P < 0.01, P < 0.05, respectively). The percentage of NKG2A⁺NKG2⁻CD8⁺ T cells was negatively correlated with CD4⁺T cell counts (r=-0.499, P < 0.01), while the percentage of NKG2D⁺NKG2A⁻CD8⁺ T cells was positively correlated with CD4⁺ T cell counts (r= 0.494, P < 0.01). The percentage of NKG2D⁺NKG2A⁻CD3⁺CD8⁻ T cells was also positively correlated with viral load (r= 0.527, P < 0.01) and negatively correlated with CD4⁺ T cell counts (r=-0.397, P < 0.05). Finally, HAART treatment reversed the changes in NKR expression caused by HIV infection. These results indicate that the expression of NKRs on T cells may be correlated with HIV disease progression.

Syphilis and HIV seroconversion among a 12-month prospective cohort of men who have sex with men in Shenyang, China.

BACKGROUND: Cross-sectional studies have found a high prevalence of syphilis and HIV infection among men who have sex with men (MSM) in China. METHODS: A total of 218 HIV-negative MSM participated in this prospective cohort study. Interviewer-administered questionnaires were completed, and blood samples were obtained for HIV and syphilis testing, both upon enrollment and at 12-month follow-up. RESULTS: Of enrolled participants, 56% (122) were retained for the full 12-month follow-up period. The cohort had an HIV incidence density of 5.4 (95% CI: 2.0-11.3)/100 person-year (PY) and a syphilis incidence density of 38.5(95% CI: 27.7-50.2)/100 PY. Having syphilis (odds ratio [OR]: 11.4, 95% CI: 1.2-104.7) and more than 5 male sexual partners within the past 12 months (OR: 6.5, 95% CI: 1.1-39.8) were independent risk factors for HIV seroconversion (each P < 0.05). Being married (OR: 3.5, 95% CI: 1.4-8.2) and having more than 5 male sexual partners within the past 12 months (OR: 4.7, 95% CI: 2.0-6.2) were risk factors for syphilis seroconversion, while age > or =30 (OR 2.1, 95% CI 0.7-9.5) and having recently engaged in unprotected receptive anal sex (OR: 2.4, 95% CI: 0.7-13.1) were marginally associated with syphilis seroconversion. CONCLUSION: The high incidence rates of HIV and syphilis in the Shenyang MSM community are significant cause for concern. The seroconversion rate for syphilis, in particular, indicates the high prevalence of high-risk sexual behaviors and the potential for increased HIV transmission. Appropriate interventions that address MSM-specific issues, including stigma, pressures from traditional society, and bisexual behavior, need to be tailored to inform and empower MSM in order to prevent HIV and syphilis in this community.

Disease-Specific Survival with Spindle Cell Carcinoma of the Head and Neck.

OBJECTIVES: (1) Determine factors influencing survival in patients diagnosed with spindle cell carcinoma (SpCC), a rare variant of head and neck squamous cell carcinoma (SCC). (2) Compare survival of patients with SpCC to those with conventional SCC. STUDY DESIGN: Retrospective cohort study. SETTING: Surveillance, Epidemiology, and End Results 18 database (years 2004-2009). SUBJECTS AND METHODS: Among patients receiving treatment for a single primary in the oral cavity, oropharynx, hypopharynx, or larynx, 118 subjects with SpCC and 18,298 subjects with SCC were identified with complete data for the variables of age, sex, grade, tumor size, stage group, and TNM stage. Disease-specific survival curves were compared. Univariate and multivariate analyses were used to examine the effects of each factor on survival over all sites and within each of 3 sites. RESULTS: Univariate analysis of the combination of the 3 anatomic subsites showed survival with SpCC was worse than with conventional SCC (P < .001). Three-year disease-specific survival with SpCC was 49.5%, and 5-year disease-specific survival was 40.2%. Compared with conventional SCC, survival was worse for SpCC of the oral cavity (P < .001) and oropharynx (P < .001) but no different for the larynx and hypopharynx site (P = .15). Multivariate analysis identified age (P = .02), tumor size (P = .006), and M stage (P < .001) as the only variables significantly affecting survival with SpCC. All variables significantly affected survival with conventional SCC. CONCLUSIONS: Spindle cell carcinoma carries a worse prognosis than SCC. Larger tumor size, older age, and metastatic disease portend worse survival with SpCC of the head and neck.

The effect of antibiotics on the microbiome in acute exacerbations of chronic rhinosinusitis.

BACKGROUND: Current treatment of acute exacerbations of chronic rhinosinusitis (CRS) is driven by identification of predominant bacteria using culture-based methods and determination of antibiotic sensitivities. The objective of this study was to evaluate the response of the sinonasal microbiome to antibiotic therapy in the setting of an acute exacerbation of CRS. METHODS: Aspirate and swab samples for culture and DNA analysis were collected bilaterally from 8 CRS patients presenting with acute exacerbations. Patients were started on a 2-week course of a culture-directed antibiotic after sensitivities were determined. Repeat samples were taken immediately on the completion of treatment. DNA was extracted from each sample, amplified using bacterial 16S primers and sequenced. Bacterial abundance was determined by quantitative polymerase chain reaction (qPCR). Diversity metrics of the microbiota between pretreatment and posttreatment samples were calculated. RESULTS: There was significantly more bacterial DNA present in the pretreatment group than in the posttreatment group. An increase in α-diversity was found in the posttreatment group relative to the pretreatment group (p < 0.05 in each comparison) with swab sampling, but not by aspirate sampling. The predominant organism identified by 16S sequencing correlated with the culture-identified bacteria genus in each patient. CONCLUSION: Significant differences exist in the diversity of bacteria populations during acute exacerbations of CRS and after antimicrobial treatment. After therapy, the increase in diversity is accompanied by a decrease in the total of abundance of the bacterial population.

Mimotopes selected by biopanning with high-titer HIV-neutralizing antibodies in plasma from Chinese slow progressors.

OBJECTIVE: One approach to identifying HIV-1 vaccine candidates is to dissect the natural antiviral immune response in treatment-naïve individuals infected for over ten years, considered slow progressor patients (SPs). It is suspected that SP plasma has strongly neutralizing antibodies (NAb) targeting specific HIV viral epitopes. METHODS: NAbs levels of 11 HIV-1-infected SPs were detected by PBMC-based neutralization assays. To investigate SP NAb epitope, this study used a biopanning approach to obtain mimotopes of HIV-1 that were recognized by SP plasma NAbs. IgG was purified from high-titer NAb SP plasma, and used as the ligand for three rounds of biopanning to select HIV-specific mimotopes from a phage-displayed random peptide library. Double-antibody sandwich ELISA, competitive inhibition assays, and peptide sequence analysis were used to evaluate the characteristics of phage-borne mimotopes. RESULTS: SPs had significantly more plasma neutralizing activity than typical progressors (TPs) (p=0.04). P2 and P9 plasma, which have highest-titer HIV-NAb, were selected as ligands for biopanning. After three rounds of biopanning, 48 phage clones were obtained, of which 22 clones were consistent with requirement, binding with HIV-1 positive plasma and unbinding with HIV-1 negative plasma. Compared with linear HIV-1 protein sequence and HIV-1 protein structure files, only 12 clones were possible linear mimotopes of NAbs. In addition, the C40 clone located in gp41 CHR was found to be a neutralizing epitope, which could inhibit pooled HIV-1 positive plasma reaction. CONCLUSION: Biopanning of serum IgG can yield mimotopes of HIV-1-related antigen epitopes. This methodology provides a basis for exploration into HIV-1-related antigen-antibody interactions and furthers NAb immunotherapy and vaccine design.

New trends of primary drug resistance among HIV type 1-infected men who have sex with men in Liaoning Province, China.

To elucidate the recent changes in prevalence of HIV-1 primary resistance mutations in men who have sex with men (MSM) in Liaoning province, 217 samples from antiretroviral therapy-naive MSM were collected. For 201 samples, the entire protease gene and 256 amino acids of the reverse transcriptase gene were successfully amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and nested PCR of viral RNA and were sequenced. Among the amplified pol sequences, HIV-1 CRF01_AE accounted for 87.6% (176/201), subtype B accounted for 8.0% (16/201), and subtype CRF07_BC accounted for 4.5% (9/201). The overall prevalence of mutations conferring resistance to any drug was 4.5%, representing 4.5% for protease inhibitor (PI)-related mutations, 0.5% for nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-related mutations, and 0.5% for nonnucleoside reverse transcriptase inhibitor (NNRTI)-related mutations. Included were V32I (0.5%), M46I (2.0%), L90M (2.0%), T215C (0.5%), and Y188L (0.5%). Only one case carried resistance mutations to all three drug classes (L90M, L10I, and A71T to PI; T215C to NRTI; and Y188L to NNRTI). L10I (4.5%), V118I/IV (17.4%), and K103R/KR (10.0%) were commonly observed mutations, but do not confer any drug resistance to PIs, NRTIs, and NNRTIs. CRF01_AE is becoming a major HIV-1 infection subtype among MSM of Liaoning province. Relatively high rates of HIV drug-resistant mutations to PIs in antiretroviral treatment-naive patients in the study represent a serious challenge for future HIV treatment programs in China.

Association of variations of NAb 2F5 and 4E10 epitopes and disease progression in Chinese antiretroviral treatment-naïve patients infected with HIV-1 clade B'.

BACKGROUND: Studies on human immunodeficiency virus type 1 (HIV-1) vaccines have recently focused on targeting the conserved neutralizing epitopes 2F5 and 4E10, and hence it is important to understand the extent of mutations in these two viral epitopes. Here, we investigated the amino acid mutations in epitopes of 2F5 (ELDKWA, HIV-1 HXB2 env 662 - 667 aa) and 4E10 (NWFDIT, HIV-1 HXB2 env 671 - 676 aa) in the membrane proximal-external region of gp41 from clade B' HIV-1-infected individuals living in Henan province, China. We also examined the frequency of a mutation and its relation to disease progression. METHODS: A cohort of 54 treatment-naïve HIV-1-infected individuals was recruited in this study, and 16 individuals were selected for a short-term longitudinal study on sequence evolution. The HIV-1 env gp41 gene was amplified, cloned, and sequenced, and predicted amino acid sequences were aligned for analysis. RESULTS: The mutations E662A and K665E on the 2F5 epitope and N671S and T676S on the 4E10 epitope were seen. Simultaneous RNA sequencing showed some discrepancies with proviral DNA sequences. In our longitudinal study, mutation levels of these two neutralizing epitopes were low but diverse and persistent. The frequencies of mutations within the 4E10 peptide NWFDIT in slow progressors were noticeably lower than those in AIDS patients (P < 0.05). CONCLUSIONS: Antigenic variation of the neutralizing epitopes 2F5 and 4E10 is limited in subtype B' infection, and that 4E10 peptide mutation is correlated with disease progression. Monitoring epitope mutations will offer useful data for development of the candidate 2F5-like and 4E10-like antibodies to prevent and treat AIDS.

Mimotopes selected by biopanning with high-titer HIV-neutralizing antibodies in plasma from Chinese slow progressors

OBJECTIVE: One approach to identifying HIV-1 vaccine candidates is to dissect the natural antiviral immune response in treatment-naïve individuals infected for over ten years, considered slow progressor patients (SPs). It is suspected that SP plasma has strongly neutralizing antibodies (NAb) targeting specific HIV viral epitopes. METHODS: NAbs levels of 11 HIV-1-infected SPs were detected by PBMC-based neutralization assays. To investigate SP NAb epitope, this study used a biopanning approach to obtain mimotopes of HIV-1 that were recognized by SP plasma NAbs. IgG was purified from hightiter NAb SP plasma, and used as the ligand for three rounds of biopanning to select HIV-specific mimotopes from a phage-displayed random peptide library. Double-antibody sandwich ELISA, competitive inhibition assays, and peptide sequence analysis were used to evaluate the characteristics of phage-borne mimotopes. RESULTS: SPs had significantly more plasma neutralizing activity than typical progressors (TPs) (p = 0.04). P2 and P9 plasma, which have highest-titer HIV-NAb, were selected as ligands for biopanning. After three rounds of biopanning, 48 phage clones were obtained, of which 22 clones were consistent with requirement, binding with HIV-1 positive plasma and unbinding with HIV-1 negative plasma. Compared with linear HIV-1 protein sequence and HIV-1 protein structure files, only 12 clones were possible linear mimotopes of NAbs. In addition, the C40 clone located in gp41 CHR was found to be a neutralizing epitope, which could inhibit pooled HIV-1 positive plasma reaction. CONCLUSION: Biopanning of serum IgG can yield mimotopes of HIV-1-related antigen epitopes. This methodology provides a basis for exploration into HIV-1-related antigen-antibody interactions and furthers NAb immunotherapy and vaccine design.