Numerous studies have examined the factors contributing to the variation in disease progression among people living with HIV-1, identifying a range of genetic, immunologic and virologic factors that interact in a complex, multifactorial dynamic. However, investigating individual susceptibility to HIV-1 infection adds an additional layer of complexity. In this observational report, we focus on the case of a man (Case-2021) who previously remained HIV-1 negative despite 35 years of self-reported high-risk sexual behavior with other men while enrolled in the MACS/WIHS Combined Cohort Study (MWCCS). To understand why this individual stayed free of HIV-1 infection for so long and what led to his eventual seroconversion, we examined the available genetic, immunologic, and virologic laboratory data before and two weeks after his diagnosis of HIV-1 infection and initiation of antiretroviral therapy (ART). In addition, we looked into two other high-risk men from same group (HR-10) who have sex with men (MSM) enrolled in the MWCCS who seroconverted later in their enrollment. Furthermore, we performed similar laboratory tests to assess the effect of seasonal variations on blood biomarkers from the same clinical visit as Case-2021, including one individual who already was living with HIV-1 and another who has remained seronegative throughout their enrollment in the MWCCS. In conclusion, this Case-2021 observational report suggests that resistance to HIV-1 infection is likely multifactorial, involving high risk sexual behavior, HIV-1 variants, the genomic structure of the CCR5 receptor on CD4+ T cells, and age-related immune changes. Further study is needed to investigate both the genomic protective factors of CCR5 against HIV-1 infection and these age-related immune changes.

INTRODUCTION:Cisgender men who have sex with men (MSM), transgender women, cisgender women and cisgender men who have sex with women only (MSW) have differential risk of acquiring Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC), the two most commonly reported bacterial sexually transmitted infections (STIs) in the USA. Similarly, MSM and transgender women have a higher number of syphilis infections in the USA than other groups. The presence of any of these three STIs is a significant risk factor for HIV acquisition and transmission, unless taking antiretroviral medication for HIV prevention (pre-exposure prophylaxis) or treatment (antiretroviral therapy). We sought to understand the prevalence rates of STIs in various vulnerable populations by anatomical site, sex and gender, which will inform the development of targeted prevention interventions and guide future updates to national testing guidelines. METHODS:Participants are enrolled in the MACS (Multicenter AIDS Cohort Study)/WIHS (Women's Interagency HIV Study) Combined Cohort Study (MWCCS)-the longest-running observational study of both people living with HIV (PLWH) and sociodemographically similar people living without HIV (PLWOH) in the world-with a total of 13 clinical research sites across the USA; participants (N=5700) complete quantitative assessments related to a variety of behavioural and health-related factors every 6 months. Regardless of whether or not they were sexually active or symptomatic/asymptomatic, all enrolled participants (ie, cisgender MSM, transgender women, cisgender women and cisgender MSW) were asked to complete surveillance testing for bacterial STIs (ie, syphilis, CT and GC). In addition to urethral samples, cisgender MSM, transgender women and cisgender MSW collected samples to test for extragenital pharyngeal and rectal CT and GC. Using at-home testing kits, participants self-collected biospecimens (ie, dried blood spots (DBS)/ microtainer of blood, urine and rectal and pharyngeal swabs) which were mailed to a central laboratory for nucleic acid amplification testing (for CT/GC) and syphilis testing using the reverse algorithm. STI results were linked to participants' HIV testing data and self-administered surveys on sociodemographics, sexual behaviours and psychosocial factors. RESULTS:Among those tested, 24.6% of cisgender MSM, 25.8% of transgender women, 7.4% of cisgender women and 14.6% cisgender MSW tested positive for at least one STI. Multiple STIs/anatomical sites were detected in 3.9% of participants, with the highest prevalence among transgender women (9.7%). Current/past syphilis prevalence was 11.2% across all participants, with cisgender MSM (19.7%) and transgender women (19.4%) having a higher prevalence than cisgender women (6.4%) and cisgender MSW (12.6%). Regarding CT, 2.1% of the participants tested were positive at one anatomical site. Among those who had extragenital testing, rectal CT was the most prevalent (3.2%), including 3.4% among cisgender MSM, 8.9% among transgender women and 0.9% among cisgender MSW. For GC, 1.4% of participants tested positive at one site. Among those who tested for extragenital STIs, rectal GC was the most common site, with a prevalence of 2.2%, including 2.3% for cisgender MSM, 4.3% for transgender women and 1.4% among MSW. Overall, 7.1% of participants tested positive for CT or GC at one or more anatomical sites. Finally, PLWH had a significantly higher prevalence of syphilis (13.74%) compared with PLWOH (6.11%) (p<0.001). CONCLUSION:There is a high prevalence of bacterial STIs among participants in the MWCCS. Self-collection of DBS/microtainer of blood, urine and rectal and pharyngeal swabs provided a useful, cost-effective option for screening participants outside of the traditional clinical setting. Given the possible asymptomatic nature of all three STIs, regular testing and education can be key to detection and treatment. Culturally appropriate and locally derived community outreach and engagement can be highly effective in reducing stigma around HIV and STIs and help reduce barriers to testing and treatment.

Objective: Lymphocyte phenotyping is a valuable tool for monitoring the effects of antiretroviral therapy on individuals living with HIV-1. A switch study was conducted to compare T-cell subset quantification performed by a research laboratory and a diagnostic, laboratory to understand the impact on the retrospective and prospective results of a long-term study. Methods: Using FACSCanto II Flow Cytometers, EDTA anticoagulated peripheral blood from 73 males enrolled in the Multicenter AIDS Cohort Study/Women Interagency HIV Combined Cohort Study was analyzed by both a research (laboratory 1) and a diagnostics laboratory (laboratory 2) for quantification of cluster of differentiation (CD)3, CD4, and CD8 T-cells. There were 47 males living with and 26 living without HIV-1. Results: Bland-Altman (B-A) analysis was applied to assess the agreement between laboratory 1 and laboratory 2 results. There were 69 out of 73 CD3, 71 out of 73CD4, and 72 out of 73 CD8 T-cell results that fell within acceptable B-A limits of agreement. The mean differences between the 2 laboratories were -1.000, -0.945, and +0.685(%), respectively. Conclusion: The strong agreement between results from laboratory 1 and laboratory 2 for CD3, CD4, and CD8 T-cell percentage suggests that the difference between laboratories using the same instrumentation and methodology will have a minimal effect on long-term study results.

PDF - 64K, Supplementary Table 4. Evaluation of EMP2 IgG1 toxicity compared to control IgG following 7 weeks of treatment delivered systemically IP. Supplementary Table 5. Evaluation of EMP2 IgG1 toxicity compared to control IgG following systemic IP treatment of up to 40mg/kg.

PDF - 54K, Supplementary Table 2. Clinical Data for Invasive Breast Cancer Patients used in the TMA.

PDF - 51K, Supplementary Table 3. Expression levels of EMP2 in triple negative breast cancer.

Introduction Interleukin 6 (IL-6) activates cells through its unique heterodimeric signaling complex of IL-6 receptor (IL6R) subunit and interleukin 6 signal transducer β-subunit glycoprotein 130 (gp130). The objective of this study was to investigate associations among serum levels of IL-6, sIL-6R, sgp130 and relative fluorescence intensity (RFI) of the α-subunit of the IL-6 receptor (CD126) on T-cells of HIV-1 infected and uninfected men. Methods Blood samples were obtained from 69 HIV-1-infected men on Highly Active Antiretroviral Therapy (HAART) with mean age of 49.1 and 52 HIV-1-uninfected with mean age of 54.3 years -. All men were participating in the Los Angeles Multi-Center AIDS Cohort Study (MACS). Serum levels of IL-6, sIL-6R, sgp130 were measured by enzyme-linked immunoassays and T-cell phenotypic analysis and RFI of CD126 on CD4 + and CD8 + by flow cytometry. Results Mean serum levels of IL-6, sIL6R, sgp130 and of CD126 RFI on CD4 + were 4.34 pg/mL, 39.3 ng/mL, 349 ng/mL and 526 RFI respectively for HIV-1-infected men and 2.74 pg/mL, 41.9 ng/mL, 318 ng/mL and 561 RFI respectively for HIV-1-uninfected men. The mean serum concentrations of IL-6, sIL-6R in HIV-1-infected and uninfected men were not significantly different (p>0.05). There was a positive correlation between plasma HIV-1 RNA and the levels of IL-6 (p<0.001), sIL6R (p = 0.002) but no correlation with sgp130 (p = 0.339). In addition, there was a negative correlation between serum levels of IL-6 with RFI of CD126 on CD4 + (p = 0.037) and a positive correlation between serum levels of sgp130 (p = 0.021) and sIL-6R in HIV-1-infected men. Conclusion Knowledge of biological variation, differences in the blood levels of biomarkers among healthy individuals and individuals experiencing illness, are very important for selection of appropriate tests for stage and progression of disease. Our data suggest no correlation among IL-6, and sIL-R6, in the treated phase of HIV-1 infection. The action and blood level of IL-6 and its receptors may be different at each stage of a disease progression.

PDF - 59K, Known Profile of EMP2 Upregulation in Breast Cancer.

PDF - 855K, Supplementary Figure 1. Anti- EMP2 IgG1 can detect EMP2 in human tissue. Supplementary Figure 2. The serum levels of the human EMP2 IgG1 was compared to those of Trastuzamab.

PDF - 68K, Supplementary Materials and Methods.

PDF - 55K, Legends for Supplementary Tables 1 through 5.

Abstract Background To assess the long-term biological coefficient of variation within individuals (CVI) and between individuals (CVG), effect of aging and cholesterol lowering drugs on blood levels of lipids in HIV-1-infected and -uninfected men. Methods Bloods were analyzed every six months over 17 years for total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in 140 HIV-uninfected (38–66 years old) and 90 HIV-treated infected (48–64 years old) white Caucasian men to examine CVI, CVG, and the effect of cholesterol lowering drugs (CLDs) on lipid levels, and estimated changes per year of biomarkers. Results With exception of HDL-C, the long term CVI compared with CVG were higher for serum levels of TC, TGs, and LDL-C in both HIV-1 infected and uninfected men not taking CLDs. Excluding results of TGs in HIV positive men, the CVI compared with CVG were lower for serum levels of TC, HDL-C, and LDL-C in both groups not taking CLDs. There were significant (p < 0.05) differences in the median serum values of lipid biomarkers among 77 HIV negative men taking and 63 not taking CLDs. Also, with exception of HDL, there were significant (p < 0.05) differences in the median values of TC, TGs and LDL-C among 28 HIV positive men taking or not taking CLDs. Conclusion Long term CVI and CVG of biomarkers will be useful for monitoring antiviral therapy side effects on lipid profiles in HIV-infected men. CVI of HIV-infected men for TC, TGs, HDL, LDL were higher significantly than CVI of HIV-uninfected men. Interestingly the long term CVI were higher than CVG for the men, who were on CLDs compared to men not on CLDs. The long-term pattern of CVI and CVG of lipid markers in both HIV-infected and uninfected men on CLDs differed from their short-term pattern.

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Background Clinicians often use population-based reference intervals (RIs) when interpreting patient results. However, this method can present problems if the analyte in question has wide variability from person to person. Methods We examined the biological variation of routine hematologic markers in 82 white non-Hispanic men every 6 months during a 30-year period, to determine the usefulness of population-based RIs and age-related decline of hematological markers. Results Many of these markers showed significant person-to-person differences (index of individuality <1.4 in 10/11 markers) and change over time with a decrease in mean for white blood cells (WBCs), red blood cells (RBCs), hemoglobin, hematocrit, platelets, and neutrophils. The mean increased for monocytes, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) (all P <.05). Conclusion Longitudinal analysis demonstrated significant decline in hematologic marker counts, with the exception of MCV and MCH. Establishment of a personalized baseline for hematologic assessments may be more useful to clinicians than previous methods.

Table S1a + b. The mean, median, and inter quartile range (IQR) values of eighteen serum biomarkers (IL-1β, IL-6, IL-1ra, IFN-γ, TNF-α, IL-8, MIP-1β, RANTES, Adiponectin, Leptin, sCD14, sCD40L, sCD163, spg130, sIL-2Rα, sIL-6R, sTNF-RII, and neopterin) and five lymphocyte phenotypes (CD3, CD4, and CD8 T-cell, CD19 B-cell, and CD56/16 NK cell) were calculated and presented for six males, six females, and both genders. (DOCX 18 kb)

Background Cytokines, chemokines, adipocytokines, soluble cell receptors, and immune activation markers play an important role in immune responsiveness and can provide prognostic value since they reflect underlying conditions and disease states. This study was undertaken to investigate the components of biological variation for various laboratory tests of blood immunological biomarkers. Results Estimates of intra-individual coefficient of variation (CV I ) and inter-individual coefficient of variation (CV G ) were examined for blood immunological biomarkers. Biomarkers with CV I < 10% for both genders were CD3, CD4, and CD8 T-cells, serum levels of soluble cluster of differentiation 14 (sCD14), sCD163, and soluble glycoprotein 130 (sgp130). The CV I for serum levels of adiponectin, interleukin-1 receptor antagonist (IL-1Ra), macrophage inflammatory protein 1 beta (MIP-1β), soluble CD40 Ligand (sCD40L), soluble interleukin-2 receptor alpha (sIL-2Rα), soluble interleukin-6 receptor (sIL-6R), soluble tumor necrosis factor receptor II (sTNF-RII), and tumor necrosis factor alpha (TNF-α) were between 11 and 20%. Biomarkers with CV G < 20% were CD3 T-cell, and serum concentrations of sCD14, sCD40L, and sgp130. The biomarkers with CV G > 40% were adiponectin, IL-1ra, leptin, MIP-1β, sCD163, and sIL-2Rα. Conclusion The biological variations of biomarkers have important monitoring value for longitudinal investigation and are essential for quality specification of tests that are performed in the laboratory. The CV I was relatively small while CV G was comparatively large and mean values of each biomarker vary between subjects. The individuality of biomarkers significantly influences reference interval values. A majority of the biomarkers in this study had strong individuality and the result of each biomarker should be cautiously interpreted if using established reference interval values. Comparison of a patient’s test result with previous ones may be more useful than the usage of conventional reference values.

Background: The use of anticoagulants may influence the composition of blood cells and interfere with plasma levels of IL-1ra when unprocessed EDTA blood samples are stored for long periods of time. Methods: Blood was drawn into EDTA and heparinized blood collection tubes from 11 HIV-1 negative men participating in the Multicenter AIDS Cohort Study (MACS) and 4 healthy volunteers. The blood was processed according to the experiments detailed in the method and after incubation; supernatants were collected and stored at -70 degrees C until batch testing using IL-1ra ELISA. Results: There was no difference between the levels of IL-1ra in EDTA blood collected into plastic and glass tubes (p = .911). There were significant increases from baseline levels of IL-1ra (p <= .05) after 24 h incubation for diluted whole blood and PBMC supernatants but not for granulocytes supernatants. Conclusion: EDTA as an anticoagulant influences the blood concentrations of IL-Ira in unprocessed blood. Thus, EDTA blood is not a suitable specimen for measurement of IL-1ra. Other types of anticoagulated blood should be processed within one hour of draw whenever measuring plasma levels of IL-1ra.

BACKGROUND:Biomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24h at room and refrigerator temperature. METHODS:Blood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20-25°C) and refrigerator temperature (4-8°C) for 0.5, 2, 4, 6, 8, and 24h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation. RESULTS:IFN-γ, sIL-2Rα, sTNF-RII and β2-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature. CONCLUSION:All the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing.

OBJECTIVE Uncontrolled HIV infection progresses to the depletion of systemic and mucosal CD4 and AIDS. Early HIV infection may be associated with increases in the concentration of MIP-3α in the blood and gut fluids. MIP-3α/CCL20 is the only chemokine known to interact with CCR6 receptors which are expressed on immature dendritic cells and both effector and memory CD8+ and CD4+ T cells. The role and prognostic value of blood levels of MIP-3α in HIV-infected individuals has yet to be described. METHODS We determined the serum levels of MIP-3α, and IFN-γ, in 167 HIV-1-infected and 27 HIV-1-uninfected men participating in the Multicenter AIDS Cohort Study (MACS). The blood biomarkers were measured using enzyme-linked immunosorbent assays (ELISA) and the cell phenotypes using flow cytometry. RESULTS Median serum levels of MIP-3α in HIV-1-infected and uninfected men was significantly different (p<0.0001) and were 21.3 pg/mL and 6.4 pg/mL respectively. The HIV-1-infected men with CD4+ T cell count <200 cells/μL showed the highest median serum MIP-3α (23.1 pg/mL). Serum levels of MIP-3α in HIV-1 infected (n=167) were negatively correlated with absolute number of CD4+ T cell (p=0.01) and were positively correlated with CD38 molecules on CD8+ T cells (p=0.0002) and with serum levels of IFN-γ (0.006). CONCLUSION Serum levels of MIP-3α concomitantly increase with plasma levels of IFN-γ, CD38 expression on CD8+ T cells, and decreased of absolute CD4+ T cells in HIV-1-infected men. A higher blood level of MIP-3α may be representation of locally high level of MIP-3α and more recruitment of immature dendritic cell at site of infection. Involvement of CCR6/CCL20 axis and epithelial cells at the recto-colonel level may enhance sexual transmission of HIV-1 in MSM and may be useful as a prognostic marker in HIV-1-infection and AIDS.

Measurement of circulating cytokine levels can provide important information in the study of the pathogenesis of disease. John L. Fahey was a pioneer in the measurement of circulating cytokines and immune-activation markers and a leader in the quality assessment/control of assays for measurement of circulating cytokines. Insights into the measurement of circulating cytokines, including consideration of multiplex assays, are presented here.