Searching for biomarkers of comorbidities in sera of treated HIV-infected patients by isoelectric focusing.

Based on their characteristics, we hypothesized that the following parameters, namely collagen IV, glutathione S-transferase, secretory component (SC), and AMP-activated protein kinase α1α2 may be useful serum markers in the detection of comorbidities in treated HIV-infected patients. These parameters were determined in 204 HIV-infected patients and 35 controls by using IEF and densitometry. Collagen IV was undetectable in controls and the majority of HIV-infected patients. Twenty-two HIV-infected patients presented significantly elevated levels of collagen IV, most of them were coinfected with hepatitis C virus and/or hepatitis B virus. SC was undetectable in controls. SC was significantly increased in 81 HIV-infected patients and significantly correlated with aspartate aminotransferase (r = 0.267, p = 0.0049), alkaline phosphatase (r = 0.309, p = 0.0011), and γ-glutamyl-transferase (r = 0.264, p = 0.0054). Glutathione S-transferase levels of HIV-infected patients were significantly higher than the controls (3779 ± 5860 vs. 785 ± 71 DU, p = 0.0007) and significantly correlated with serum urea (r = 0.204, p = 0.0038), triglycerides (r = 0.209, p = 0.0033), and lipase (r = 0.219, p = 0.0025). AMP-activated protein kinase α1α2 levels of HIV-infected patients were significantly higher than the controls (5676 ± 6248 vs. 1189 ± 6248 DU, p = 0.0009). Further studies are needed to demonstrate the relevance of these results to diagnose non-AIDS-related illnesses in HIV-infected patients.

Serum hepcidin levels in women infected with HIV-1 under antiviral therapy.

Accumulating data suggest that iron may have a role in the regulation of HIV-infection. In the present study, we determined by radioimmunoassay the levels of hepcidin, a key regulator of iron homeostasis, in sera of 182 women infected with HIV-1 under highly active antiretroviral therapy (HAART). In the total cohort, hepcidin levels were lower in individuals infected with HIV than in controls (3.20 ± 3.06 vs. 5.68 ± 3.66 nmol/L, P = 0.009). Serum hepcidin concentrations were strongly correlated positively with iron, ferritin, urea, and uric acid. In the total cohort of patients with abnormal viral load and CD4 cell count <500 cells/mm(3) , a strong positive correlation was found between hepcidin and viral load. Hepcidin level was significantly higher in HIV-patients with high viremia than in patients with undetectable viral load. Iron level was significantly lower in HIV-patients with high viral load compared with patients with undetectable viral load. This study suggests that hepcidin controls serum iron, especially in response of iron utilization by HIV for viral replication. The possibility of using inhibitors of hepcidin expression as adjunct therapy for HIV-patients is discussed.

Detection of AMP-activated protein kinase α1 by isoelectric focusing in sera of patients with metabolic diseases.

The aim of this study is to determine by isoelectric focusing the level of AMPKα1, an energy sensor, in sera of patients who are in energy-demanding situation. After Western blotting, detection was performed with specific antibodies against AMPKα and its phosphorylated form. To evaluate the effect of weight loss on AMPK, sera from 24 patients were collected before and after intragastric balloon insertion over a 16-week follow-up period. Compared to baseline, all patients showed postoperatively an increase of AMPK. Patients with ischemic heart, with inflammatory bowel disease, with chronic undernutrition or with hepatic diseases were examined. Compared to control subjects, the majority of them showed a significant increase of AMPK. These results suggest that serum AMPK may have a potential for diagnosis of several metabolic diseases. However, this has to be confirmed by further studies with additional biomarkers and with more specific techniques.

Soluble chemokine receptor CXCR4 is present in human sera.

A soluble form of the chemokine receptor CXCR4 was detected in human sera by isoelectric focusing and Western blotting. Sera of patients and normal subjects were analyzed using a panel of specific antibodies. Compared with controls, high levels of serum CXCR4 were found in patients with inflammatory bowel diseases. Serum CXCR4 levels in the majority of HIV patients were similar to those in healthy controls. A sensitive polyclonal antibody was developed in rabbit immunized with a maltose binding protein (MBP) construct expressing the full-length CXCR4. Using anti-MBPCXCR4 antibody, the level of CXCR4 in sera of a majority of patients with fibrosis was very low. The potential of serum CXCR4 as a new diagnostic biomarker warrants further investigation.

Tolerance and efficacy of an air-filled balloon in non-morbidly obese patients: results of a prospective multicenter study.

BACKGROUND: Intragastric balloons have been proposed to induce body weight loss in obese subjects. Most studies were performed using liquid-filled balloons. Air-filled balloons may increase digestive tolerance. Our goal was to study the tolerance and efficacy of a new air-filled intragastric balloon in nonmorbidly obese patients. METHODS: 32 patients were included, with a mean BMI of 35.0 (range 30.1-40.0). The balloon was inserted under general anaesthesia, inflated with 800 ml of air, and removed 4 months later. Tolerance and body weight were monitored until 12 months after removal. Ghrelin levels were measured before balloon insertion, 1 and 4 weeks after, and before removal. RESULTS: Weight loss was significant at 1, 2 and 4 months after balloon insertion (6, 7 and 10 kg, respectively, P<0.001). Early removal of the balloon occurred in 3 cases. 28 patients were contacted 12 months after balloon removal: 2 had undergone gastric banding; among the 26 remaining, the mean weight loss was 7 kg. 9 patients (30%) remained with a weight loss >10%, and satisfaction with the method was 87% for these 9 patients, and 22% for the other patients who had weight loss <10% (P<0.04). Fasting plasma ghrelin levels increased at week 1 and 4 after balloon insertion, and decreased at week 16 (P<0.001). CONCLUSIONS. The air-filled intragastric balloon was safe. Its effect on weight loss appeared equivalent to other balloons. 12 months after balloon removal, 30% of the patients maintained a weight loss >10%.

Hyperglycosylated ferritin in sera of HIV-1-infected patients treated with highly active antiretroviral therapy.

A study was conducted to determine the relationship between ferritin and glycosylated isoforms of ferritin and insulin resistance in 69 HIV-infected men receiving HAART. Ferritin levels were significantly correlated with aspartate aminotransferase, alanine aminotransferase, bilirubin and with insulin resistance. The ferritin isoelectric focusing patterns of five insulin-resistant HIV-infected patients under HAART showed large amounts of hyperglycosylated isoforms, which was not found in 56 control subjects and 46 untreated HIV-1-infected patients.

Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups.

A soluble form of the HIV-1 envelope glycoprotein gp160 devoid of the transmembrane anchor domain was found to bind to cholesteryl-hemisuccinate agarose. The external subunit gp120 failed to bind to the resin, suggesting that the site responsible for the binding to cholesterol was located in the transmembrane protein gp41. We constructed a series of maltose binding protein (MBP) fusion proteins representing overlapping fragments of the gp41 molecule and we studied their capacity to bind to cholesteryl beads. The domain responsible for binding to cholesterol was localised within the residues 668 to 684 immediately adjacent to the membrane spanning domain. We identified a short sequence (LWYIK, aa 678-683) comparable to the cholesterol interaction amino acid consensus pattern published by Li and Papadopoulos [Endocrinology 139 (1998) 4991]. We demonstrated that the sequence LWYIK synthesized fused to the MBP was able to bind to cholesteryl groups. A synthetic peptide containing the sequence LWYIK was found to inhibit the interaction between cholesteryl beads and MBP44, an MBP fusion HIV-1 envelope protein that contains the putative cholesterol binding domain. Human sera obtained from HIV-1 seropositive patients did not react in ELISA to the LWYIK sequence, suggesting that this region is not exposed to the immune system. The biological significance of the interaction between gp41 and cholesterol is discussed.

Effects of intragastric balloon on gastric emptying and plasma ghrelin levels in non-morbid obese patients.

BACKGROUND: Intragastric balloons have been proposed to induce weight loss in obese subjects. The consequences of the balloon on gastric physiology remain poorly studied. We studied the influence of an intragastric balloon on gastric emptying and ghrelin secretion in non-morbid obese patients. PATIENTS AND METHODS: 17 patients were included in the study, with mean BMI of 34.4 (range 30.1-40.0). The balloon was inserted under general anaesthesia and endoscopic control, inflated with 600 ml saline, and removed 6 months later. Body weight and gastric emptying (13C-octanoic acid breath test) were monitored while the balloon was in place and 1 month after removal. Ghrelin levels were measured just before balloon insertion and removal. RESULTS: Mean weight loss was 8.7 kg (range 0-21). Gastric emptying rates were significantly decreased with the balloon in place, and returned to pre-implantation values after balloon removal. Plasma ghrelin levels were significantly decreased (95% CI: -3.8 to -20.7 ng/ml), despite concomitant weight loss. Weight reduction was not correlated to the effect of the balloon on gastric emptying, but was significantly correlated to the ghrelin variations (r=0.668, 95% CI: 0.212-0.885). CONCLUSIONS: Gastric emptying rates and plasma ghrelin levels are decreased in the presence of intragastric balloon. Weight loss induced by the intragastric balloon is related to ghrelin variations, but not to gastric emptying. Ghrelin inhibition may explain part of the effect of the balloon on satiety.

Leptin, adiponectin, and ghrelin dysregulation in chronic kidney disease.

In the past 10 years, 3 new metabolic compounds, leptin, adiponectin, and ghrelin, involved in energy metabolism, body composition, and appetite regulation, have been discovered. We have assessed their characteristics in 46 patients with stage 3 to 4 chronic kidney disease to evaluate the role of decreased renal function in the abnormal handling reported in more severe end-stage renal disease patients. In addition to the usual correlations with body mass index and body fat mass, the results show unexpected positive correlations between leptin and insulin, leptin and adiponectin, a weak inverse relationship between adiponectin and glomerular filtration rate, and no influence of C-reactive protein on either leptin or adiponectin in these noninflamed patients. Serum ghrelin was inversely correlated with body mass index and with glomerular filtration rate as measured by inulin clearance. Thus, ghrelin and leptin, 2 antagonist signals for energy balance, both seem to increase when glomerular filtration rate is reduced, potentially neutralizing their respective biologic effects in severe renal insufficiency.

Detection of thyroglobulin in antithyroglobulin antibody-positive sera by isoelectric focusing.

Circulating antibodies have the potential to interfere with the measurement of thyroglobulin (Tg) in sera of patients. Here, we determined Tg concentration by isoelectric focusing (IEF) on agarose gel using for detection a rabbit antiserum to human Tg termed FLX. Tg was determined in sera of thyroid patients and HIV-infected patients under antiviral therapy. We showed that Tg IEF was not affected by the presence of anti-Tg antibodies (TgAb). Tg concentrations measured by IEF in TgAb-negative sera were in most of the cases, similar to those obtained by IRMA (immunoradiometric assay). However, in 5 of the 96 thyroid patients, and none of the 46 healthy subjects, Tg was undetectable by antiserum FLX and measurable by IRMA. In HIV-infected patients (64 men and 60 women), Tg was not recognized by FLX in 23 men and 9 women and this was related to abnormal CD4. We hypothesize that the decreased binding of FLX to Tg may be the result of conformational change on the Tg molecule, a phenomenon apparently related to immunodeficiency in HIV-infected patients. For thyroid patients, Tg IEF may be very useful for the interpretation of results when Tg measurements by IRMA and automated immunoassays are affected by interferences.

Detection of IgA inhibiting the interaction between gp120 and soluble CD4 receptor in serum and saliva of HIV-1-infected patients.

OBJECTIVE: To evaluate the presence of IgA directed to the CD4-binding domain of gp120 and to a conserved region of gp41 (the Kennedy epitope) in serum and parotid saliva of HIV-1-seropositive patients. METHODS: IgA were separated from IgG by anion-exchange chromatography and protein G treatment. The reactivity of IgA was tested against peptides and fusion proteins of the maltose-binding protein (MBP) and the CD4-binding site (MBP24) and MBP and the Kennedy epitope (MBP42). The capacity of serum and saliva IgA to interfere with the gp120-soluble CD4 (sCD4) interaction was examined. IgA were also purified by affinity chromatography using the MBP proteins adsorbed to a resin. RESULTS: Peptides representing the CD4-binding domain and the Kennedy epitope were recognized by serum and saliva IgA of HIV-1-seropositive patients. Of the sera and saliva samples tested, 6/26 serum IgA and 5/25 saliva IgA inhibited the gp120-sCD4 interaction by approximately 50%. The gp120-sCD4 interaction was inhibited by MBP24 affinity-purified IgA but not by MBP42 affinity-purified IgA. CONCLUSION: Immunogens capable of eliciting IgA antibodies that inhibit gp120-CD4 binding might be efficiently used in vaccine to prevent mucosal transmission of HIV-1.

Glucose-to-insulin ratio rather than sex hormone-binding globulin and adiponectin levels is the best predictor of insulin resistance in nonobese women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS), the main androgen disorder in women, has been suggested to be associated with a high risk of developing cardiovascular disease and type 2 diabetes. In many PCOS patients, overweight or central obesity is generally associated with increases in fasting insulin levels, insulin resistance, and glucose intolerance, and has been identified as a target for new therapeutic strategy, including early change in lifestyle. Early biochemical marker(s) for identifying at-risk patients will be useful for prevention studies. The main goal of the present study was to search for such tool(s). We investigated 16 nonobese PCOS women by performing euglycemic hyperinsulinemic clamp and measuring insulin levels during fasting and oral glucose tolerance test, as well as the serum concentrations of SHBG, leptin, and adiponectin, the newly identified adipose factors. Eight of the 16 patients had a steady-state glucose disposal rate less than 8.5 mg/kg.min, the lowest normal value for nonobese control women. These insulin-resistant patients had significant higher body mass index (BMI) and waist-to-hip ratio (WHR), and lower high-density lipoprotein cholesterol and SHBG levels. As expected, glucose disposal correlated negatively with BMI (P = 0.01), WHR (P = 0.01), and fasting insulin level (P = 0.003). On stepwise regression analysis, however, the glucose-to-insulin ratio (GIR) emerged as the strongest independent parameter to appraise insulin resistance (R(2) = 0.61). SHBG level correlated positively with GIR (P < 0.001) and negatively with BMI (P = 0.003) but did not correlate with either insulin response during the glucose tolerance test or plasma leptin and/or adiponectin levels. In contrast, BMI was the only independent predictive parameter of SHBG (P = 0.003, R(2) = 0.73). Interestingly, plasma adiponectin levels were positively associated with glucose disposal rate (P = 0.043) and negatively with WHR (P = 0.024), waist circumference being the best predictor of adiponectin level (P < 0.01). Leptin level correlated only with BMI (r = 0.62, P = 0.01). This study confirmed that insulin resistance, despite the lack of obesity as such, is clearly present in many PCOS women, and demonstrated that GIR is the best predictor for insulin resistance. It was also shown that adiponectin level is a good indicator of abdominal fat mass and is associated to insulin resistance. Finally, low SHBG levels in PCOS are intimately associated with BMI, suggesting that some signal(s) from the adipose tissue, independent of adiponectin and leptin, may regulate liver production of SHBG.

Ability of antibodies specific to the HIV-1 envelope glycoprotein to block the fusion inhibitor T20 in a cell-cell fusion assay.

The anti-HIV peptide T20 is able to inhibit the syncytia formation between CHO-WT and HeLa CD4(+)cells. We found that several sera of HIV-infected patients have the capacity to block the inhibition of fusion by T20. Suggesting that these sera may contain antibody which can block T20 access and prevent membrane fusion, we studied the ability of a panel of antibodies directed to different regions of HIV-1 envelope glycoprotein to block the inhibition of fusion by T20. We found that the C1 and V3 loop regions of gp120 and the heptad repeat 1, the immunodominant C-C region and the Kennedy epitope of gp41 located in the intracytoplasmic tail were the target for antibodies capable to block the inhibition of syncytia formation by T20. We suggest that these antibodies have the capacity to counteract the anti-fusion effect of T20 by preventing its binding to the interaction sites. Further studies are needed to determine if some of them recognize new T20 interaction sites.

Detection of AMP-activated protein kinase in human sera by immuno-isoelectric focusing.

AMPKalpha is a subunit of AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that works as a fuel sensor activated in response to the depletion of cellular ATP. AMPKalpha is considered as a master switch in regulating glucose and lipid metabolism. Determining its presence in patient sera may help in diagnosing metabolic diseases. Using isoelectric focusing and Western blotting, we were able to detect AMPKalpha in human sera. Using specific antibodies, we showed that the AMPKalpha1 and alpha2 isoforms were apparently present in equal amounts in human sera. To characterize normal and abnormal AMPKalpha patterns, we used an antibody which recognized both isoforms (alpha1 and alpha2) to analyze sera of patients and healthy individuals. We also analyzed sera of HIV patients because several studies suggest that AMPK may play a role in the mechanism of lipodystrophy in HIV patients under antiretroviral therapy. We found that patients with type 2 diabetes or liver diseases presented abnormal AMPK IEF patterns. AMPK was poorly detectable in sera of patients with end-stage liver disease. Abnormal AMPK IEF patterns were more frequent in treated HIV-patients compared to those who are untreated suggesting a possible association between AMPK and the side-effects of antivirals. Our findings highlight the potential of serum AMPK as a new diagnostic biomarker and may help to study the regulation of AMPK activity in tissues.

Antibodies purified from sera of HIV-1-infected patients by affinity on the heptad repeat region 1/heptad repeat region 2 complex of gp41 neutralize HIV-1 primary isolates.

OBJECTIVE: The objective of this paper was to evaluate the presence and the neutralizing activity of antibodies directed against the complex formed between the two heptad repeat regions (HR1 and HR2) of HIV-1 gp41 in sera of HIV-1-infected patients. RESEARCH DESIGNS AND METHODS: The HR1 region was represented by the peptide N36 and the maltose-binding protein (MBP)-HR1, the HR2 region by the peptide C34 and MBP44. Antibodies directed to the HR1/HR2 complex were purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed onto a resin. RESULTS: First, we demonstrated that human monoclonal antibodies, which are directed specifically to the HR1/HR2 complex recognized in enzyme-linked immunosorbent assay the MBP-HR1/C34 and MBP44/N36 mixtures but not the proteins or the peptides individually. We investigated the ability of 50 sera of HIV-1-infected patients to react with the MBP-HR1/C34 and MBP44/N36 complexes. We found that the majority of sera of HIV-1-infected patients recognized the HR1/HR2 complexes but not or to a lower extent the proteins or the peptides individually. Antibodies purified from sera by affinity chromatography using MBP-HR1/C34 adsorbed to a resin neutralized different primary HIV-1 isolates. CONCLUSION: The presence of antibodies directed to the HR1/HR2 complex in sera of HIV-infected patients highlights the immunogenic character of the complex, whereas the neutralizing activity of these antibodies suggests that immunogens representing HIV-1 HR1/HR2 complexes might be used in anti-HIV vaccine.

Depletion in antibodies targeted to the HR2 region of HIV-1 glycoprotein gp41 in sera of HIV-1-seropositive patients treated with T20.

The anti-HIV drug T20 is a synthetic peptide derived from the HR2 region of HIV-1 gp41. T20 contains the sequence ELDKWA, which binds the broadly neutralizing antibody 2F5. Using plates coated with T20 or with synthetic peptides and recombinant proteins representing gp120 or gp41 domains, this study investigated by enzyme-linked immunosorbent assay the levels of antibodies directed to the gp160 molecule in patients treated with T20. Analysis of sera obtained before and after administration of T20 indicated that the levels of antibodies directed to T20, to MBP44, a maltose binding protein representing the HR2 region, and to 4765, a synthetic peptide containing the sequence ELDKWA, fell following administration of T20, while the levels of antibodies directed to other regions of gp41 ectodomain and to gp120 remained stable. The decline observed was independent of the viral load and of the total IgG concentration. Follow-up studies with sera obtained from HIV-1-seropositive patients naive to T20 indicated no decline in the level of antibodies directed to HR2 and other regions of gp160. Analysis of sera obtained from a patient after 2 months of T20 treatment interruption showed a level of antibodies to the HR2 region similar to that measured before administration of T20. The addition of increasing amounts of T20 to sera from T20-naive patients decreased the level of serum antibodies against peptide 4765, T20, and MBP44. The observation of antibody depletion by T20 suggests that anti-gp41 antibodies may interfere with T20 treatment by forming T20-antibody complexes.