Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum.

Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

Cerebrospinal Aß11-x and 17-x levels as indicators of mild cognitive impairment and patients' stratification in Alzheimer's disease.

In the present work, the concentrations of Aß11-x and Aß17-x peptides (x=40 or 42), which result from the combined cleavages of ß-amyloid precursor protein (AßPP) by ß'/α or α/γ-secretases, respectively, were assessed in cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease (AD) or mild cognitive impairment (MCI). Specific multiplexed assays were set up using new anti-40 and anti-42 monoclonal antibodies (mAbs) for the capture of these N-truncated Aß peptides and anti-11 or anti-17 mAbs for their detection. The specificity, sensitivity and reproducibility of such assays were assessed using synthetic peptides and human cell models. Aß11-x and Aß17-x were then measured in CSF samples from patients with AD (n=23), MCI (n=23) and controls with normal cognition (n=21). Aß11-x levels were significantly lower in patients with MCI than in controls. Compared with the combined quantification of Aß1-42, total Tau (T-Tau) and phosphorylated Tau (P-Tau; AlzBio3, Innogenetics), the association of Aß11-40, Aß17-40 and T-Tau improved the discrimination between MCI and controls. Furthermore, when patients with MCI were classified into two subgroups (MCI ≤1.5 or ≥2 based on their CDR-SB (Cognitive Dementia Rating-Sum of Boxes) score), the CSF Aß17-40/Aß11-40 ratio was significantly higher in patients with CDR-SB ≤1.5 than in controls, whereas neither Aß1-42, T-Tau nor P-Tau allowed the detection of this subpopulation. These results need to be confirmed in a larger clinical prospective cohort.

The placenta contributes to activation of the renin angiotensin system in twin-twin transfusion syndrome.

The renin-angiotensin system (RAS) in twin-twin transfusion syndrome (TTTS) is up-regulated in the donor fetus's kidneys, but down-regulated in the recipient's. Ultrasonographic and echocardiographic features suggest that the recipient is also exposed to RAS components. In this study we investigated the role and origin of RAS components in the recipient fetus. Monochorionic diamniotic (MCDA) pregnancies were recruited from a tertiary fetal medicine service. Cord blood was collected from MCDA twins (TTTS and control non-TTTS) at delivery for renin and angiotensin II immunoassays. Placental tissue was flash-frozen for mRNA and protein expression or formalin-fixed for immunohistochemistry. Archival placenta and kidney samples were used for immunohistochemistry and in-situ hybridization. Plasma renin levels were elevated (p<0.05) in recipients (median 201 pg/ml, range 54-315 pg/ml) and donors (125 pg/ml, 25-296) with TTTS compared to controls (2.5 pg/ml, 1.1-1.5 pg/ml). The same was found with angiotensin II with high levels in both recipients (300.5 pg/ml, 86.1-488 pg/ml) and donors (239 pg/ml, 76.6-422) compared to controls (169.5 pg/ml, 89-220 pg/ml, p<0.05). Renin mRNA expression, and protein appeared qualitatively higher in the placental territory of the recipient compared to that of the donor and non-TTTS controls. We conclude that both fetuses in TTTS are exposed to high levels of RAS components; these appear to be produced from different sites, namely the kidney of the donor, and the placenta of the recipient. Given the markedly different phenotypes in the genetically identical fetuses with TTTS, we suggest that the source of RAS components may influence their clinical manifestations.

Inflammatory cytokines in juvenile idiopathic arthritis: effects on physical growth and the insulin-like-growth factor axis.

OBJECTIVE: To investigate the relationship between markers of inflammation with physical growth and systemic markers of GH secretion in JIA. DESIGN: This is a cross sectional prospective study of patients with JIA recruited during therapeutic arthrocentesis of 17 children with JIA (F,10): 8 oligoarticular (OJIA) and 9 polyarticular (PJIA). RESULTS: Median adjusted height (AHt) SDS was -0.3 (-2.2 to 1.6). Serum ALS SDS (median -1.3, range -2.7 to -0.6) was reduced compared with serum IGFBP-3 SDS (median -0.5, range -7.7 to 2.3) and IGF-1 SDS (median -0.2, range -0.5 to 0.5). Log serum IL5 (95% CI -3.25, -0.81) and log serum IL15 (95% CI -9.58, -4.10) were independent factors associated with AHt SDS. Inflammatory cytokines individually showed no association with IGF-1, IGFBP-1, -2, -3 and ALS. CONCLUSION: Children with JIA and mild degree of growth retardation show decreased ALS and IGFBP-3. Cytokines did not show an association to systemic markers of GH secretion. However, this study reports the novel, preliminary association between serum levels of IL5 and IL15 and the extent of short stature.

Stage I twin-twin transfusion syndrome: rates of progression and regression in relation to outcome.

OBJECTIVES: Twin-twin transfusion syndrome (TTTS) results in high rates of perinatal mortality and neurological morbidity. Fetoscopic laser ablation of placental anastomoses is now established as the treatment of choice for advanced disease. However, there remains controversy about its use in early-stage TTTS, in which laser-related fetal losses need to be balanced against relatively favorable outcomes with more conservative approaches. We investigated rates of progression and regression in Stage I TTTS and determined factors influencing the course of the disease. METHODS: We undertook a retrospective observational study of all TTTS cases referred to our tertiary referral fetal medicine service from 2000 to 2006. In patients presenting with Stage I TTTS, the following variables were evaluated for their ability to predict the course and progression of the disease: gestational age (GA) at presentation, amniotic fluid index, recipient and donor deepest vertical pool, presence of artery-artery anastomoses, small-sized bladder compared to normal donor bladder and fetal size discordance. Study end-points were disease regression or progression, and neonatal survival at 28 days. RESULTS: Among 132 consecutive cases of TTTS, 46 women presented with Stage I disease. In the majority (69.6%), disease remained stable (28.3%) or regressed (41.3%). Of cases that progressed, 79% did so within 2 weeks and 93% progressed to at least Stage III. No factor was significantly linked with progression or regression, although there was a trend towards the absence of an artery-artery anastomosis (P = 0.10) and the presence of a small rather than normal donor bladder (P = 0.10) influencing progression, and later GA at presentation (P = 0.07) influencing regression. At least one infant survived in 83% of cases and there was double survival in 59%. Perinatal outcome was significantly better in cases that regressed (the rates of at least one survivor and double survival being 89% and 89%, respectively) or remained Stage I (77% and 61%, respectively), compared with those cases that progressed (79% and 14%, respectively). Treatment with amnioreduction at first presentation did not influence progression or regression. CONCLUSIONS: This study demonstrates that a high percentage of Stage I TTTS cases regress or remain early stage. Identification of factors predicting progression would facilitate the selection of patients for definitive therapy, while avoiding treatment-related morbidities in mild or transient disease.

Cytokine profiling and in vitro studies of murine bone growth using biological fluids from children with juvenile idiopathic arthritis.

OBJECTIVE: Growth retardation in children with chronic inflammatory disease may be partly due to direct effects of pro-inflammatory cytokines on the growth plate and requires further investigation. DESIGN: This study assessed the cytokine concentrations in serum and synovial fluid (SF) in juvenile idiopathic arthritis (JIA), and determined the effect of the biological fluid on cultured murine metatarsal growth. PATIENTS: Serum and SF were obtained from four children attending for arthrocentesis (child A, systemic; children B, C and D, oligoarticular). In addition, serum samples were obtained from four more children (children E and F, polyarticular; child G, oligoarticular). MEASUREMENTS: Anthropometry, cytokine levels and longitudinal bone growth were assessed. RESULTS: Cytokines were elevated to a variable extent in the samples. Although all serum samples were associated with reduced metatarsal growth, only SF from child A and child B reduced metatarsal growth. Metatarsals treated with child A's SF showed reduced proliferation, reduced proliferative and mineralizing zone width, and increased hypertrophic zone width. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 concentrations were elevated in child A's SF. However, SF exposure with neutralizing antibodies to these cytokines or IGF-1 did not improve metatarsal growth. CONCLUSION: SF and serum JIA samples can impair bone growth at the growth plate. In synovial fluid, the effect is variable but resistant to treatment with IL-1beta, IL-6 and TNF-alpha specific antibodies and IGF-1, suggesting that other factors in this biological fluid may also have an effect on longitudinal growth through IGF-1-independent mechanisms.

A randomised controlled trial of morphine versus phenobarbitone for neonatal abstinence syndrome.

BACKGROUND: The incidence of neonatal abstinence syndrome (NAS) has increased 10-fold over the last decade in Glasgow. In the Princess Royal Maternity Hospital, it now accounts for 17% of special care baby unit (SCBU) admissions. OBJECTIVE: To compare opiate replacement therapy (morphine sulphate) with the present standard treatment (phenobarbitone) for management of NAS. The primary study end point was duration of pharmaceutical treatment. Secondary end points were the requirement for additional drugs and the requirement for SCBU admission. DESIGN: Double blind, randomised controlled clinical trial. METHODS: Differential diagnoses were excluded, and two consecutive Lipsitz scores > 4 defined NAS requiring treatment. Infants were randomised to receive morphine sulphate or phenobarbitone. Treatments were identical in appearance, odour, and volume. Increments, decrements, and discontinuation of treatments were protocol driven. RESULTS: Seventy five infants participated. All mothers received opiate replacement therapy (methadone) during pregnancy and most used other drugs (n = 62, 83%). No significant difference in maternal drug use patterns was observed between treatment groups. Median treatment duration was four days shorter with opiate replacement (8 v 12 days, Mann-Whitney U test, p = 0.02). Phenobarbitone treated infants tended to require second line treatment (47% v 35%, chi(2) test, p = 0.11) and SCBU admission (62% v 30%, chi(2) test, p = 0.04) more often. CONCLUSIONS: Opiate replacement therapy appears to be superior for management of symptomatic NAS when maternal opiate use is prevalent. The shorter treatment duration and lower requirement for higher intensity nursing may have significant cost implications. Tailoring NAS treatment to local maternal drug use may result in similar benefits.

New polyazamacrocycle-nucleoside conjugates: synthesis, anti-HIV evaluation and interaction with CXCR-4 coreceptor.

We report the synthesis of new conjugates that incorporate in their structure bis-tetraazamacrocycle coupled with AZT via enzymolabile bond. Two series of bis-polyazamacrocycles-AZT conjugates were designed, synthesized and evaluated for their antiviral effect in vitro as well as their capability to bind to CXCR-4 coreceptor.

Rationale for a vaccine using cellular-derived epitope presented by HIV isolates.

It has been clearly demonstrated that cellular antigens (HLA, beta 2-microglobulin) are incorporated at the virion surface. The same epitope derived from beta 2-microglobulin is presented on all virus isolates. The peptide was identified by blocking the neutralizing capacity of a monoclonal antibody directed to R7V epitope: using this peptide for developing an ELISA, we have detected antibodies in nonprogressor patients with neutralizing property to laboratory strains and primary isolates. Purified anti-R7V antibodies immunoprecipitate all HIV isolates at concentration dependent. R7V is immunogenic after rabbit immunization and induces HIV immunoprecipitating and neutralizing antibodies. The patient's as well as the immunized rabbit antibodies did not bind to any cell. No autoimmune disease is found in nonprogressor patients. For all these reasons, R7V is a good candidate for an universal AIDS vaccine.

A novel epitope R7V common to all HIV-1 isolates is recognized by neutralizing IgG found in HIV-infected patients and immunized rabbits.

We have previously reported that the presence of antibodies (Ab) directed to the beta2-microglobulin-derived peptide R7V in patient's serum correlated with the nonprogression to AIDS. In order to investigate whether R7V motif could represent a potential target for neutralization, we have immunopurified anti-R7V Ab from sera of nonprogressors, as well as from sera of rabbits injected with R7V. We showed that human as well as rabbit purified,anti-R7V IgG precipitated laboratory adapted strains, as well as primary isolates from different clades indicating that: (1) R7V epitope is a common motif presented at the surface of genetically divergent HIV-1 strains (2) R7V is immunogenic in vivo. When used in neutralizing assay, purified anti-R7V Ab from human or rabbit origin were shown to neutralize infection by HIV-1 laboratory adapted strains and HIV-1 primary isolates. All together, our results indicate that the R7V motif shared by all HIV strains could be considered as a possible candidate for an HIV vaccine.

New bicyclam-AZT conjugates: design, synthesis, anti-HIV evaluation, and their interaction with CXCR-4 coreceptor.

We report the synthesis of mono- and bis-tetraazamacrocycle-AZT conjugates. All new compounds were screened for their ability to inhibit HIV-1 replication in MT4 cell line and were compared to AZT alone. It appears that N-protected covalent prodrugs are equipotent to AZT as inhibitor of HIV replication, while N-deprotected analogues exhibit both higher activity and selectivity against HIV-infected cells. The most active antiviral compounds 27, 28, 34, and 35 were then tested for their binding capability to CXCR-4 receptor. N-Boc analogues 27 and 34 were only weakly effective; in contrast, N-deprotected conjugates 28 and 35 were antagonists to 12G5 mAb binding until 0.05 and 5 microg/mL, respectively. The stability of compound 28 in human plasma was evaluated, and half-life was found to be approximately 8 h in the described conditions. All these results seem to demonstrate the confidence of our prodrug approach, with analogue 28 emerging as the best candidate as lead compound in HIV-1 polytherapy perspective.

Neonatal abstinence syndrome at Glasgow Royal maternity hospital.

OBJECTIVE: To get a clearer idea of the magnitude of the problem of Neonatal Abstinence Syndrome and, in particular, the number of cases and the severity and length of any withdrawal symptoms. DESIGN: Retrospective review of casenotes. SETTING: GRMH, Rottenrow Neonatal Unit Jan 1994-Aug. 1996. SUBJECTS: Infants admitted to the Neonatal Unit with the label "Infant of a Drug Abusing Mother" and their mothers. RESULTS: Sixty-four infants qualified for the audit i.e. on average two children per month. Most mothers had a polydrug habit taking both opiates and benzodiazepines. Thirty per cent of infants were premature and, as a group they were lighter and had a smaller OFC than the national average. Fifty-three infants suffered NEONATAL ABSTINENCE SYNDROME, 50 of these being "jittery, restless or irritable". Only four had convulsions. Most infants' withdrawal presented within 72 hours (median 24 hours) and lasted for less than six days. Infants exposed to methadone had symptoms for longer but required less treatment than infants exposed to heroin. Forty infants needed treatment with phenobarbitone for a median of four days. Only six infants needed other drugs. Median age of discharge was 11 days and 49 out of 53 went home with their mothers. CONCLUSIONS: The Glaswegian polydrug pattern would make morphine/methadone unsuitable treatment. If these infants are not showing signs of withdrawal at 72 hours it is, in our opinion, reasonable to discharge them. Infants should not be discharged with outstanding problems, as follow-up is usually unsuccessful.

Role of Kaposi's sarcoma cells in recruitment of circulating leukocytes: implications in pathogenesis.

OBJECTIVE: We sought to identify and characterize mechanisms of interaction between Kaposi's sarcoma cells and circulating leukocytes leading to leukocyte migration into the lesion. STUDY DESIGN/METHODS: By using static and dynamic adhesion models, we measured the ability of late-stage KSY1 cells to support adhesion and transmigration of peripheral blood lymphocytes (PBL). RESULTS: We showed that resting as well as TNF-alpha- or PMA-activated KSY1 cells supported adhesion and transmigration of PBL with a higher efficiency compared with normal endothelial cells. The LFA1/ICAM1 pathway was totally involved in PBL adhesion to resting or TNF-alpha-activated KSY1 cells and partially responsible for adhesion to PMA-activated KSY1 cells. No inhibition of adhesion was observed by blockage of the VLA4 pathway. Under flow conditions, PBL/KSY1 cell interaction was totally dependent on L-selectin. CONCLUSION: Our data indicate that KS cells mimic an endothelium-like structure by regulating extravasation of lymphocytes into lesions.

HIV as the cause of AIDS and associated diseases.

This brief article is in response to the previous article by Peter Duesberg and David Rasnick titled 'The AIDS Dilemma: Drug Diseases Blamed on a Passenger Virus.' We focus our response on thirteen specific issues raised by Duesberg and Rasnick.

[HIV virus detection in ejaculates collected at different times in seropositive patients]. / Recherche du virus VIH dans des éjaculats recueillis à des temps variables de sujets séropositifs.

Ejaculates from 22 seropositive males were collected at various times 1 to 11 ejaculates for each in one year period of time. After fractionation, no virus is detected in mobile and living spermatozoa. Moreover, HIV is not always found in all ejaculates from the same patient.

[Study of HIV localization in sperm]. / Etude de la localisation du VIH dans le sperme.

The aim of the study was to investigate the presence and the localization of HIV in human ejaculate and its different components. Sixty-three ejaculates from 19 HIV-positive patients have been studied. By using cellular as well as molecular biology methods, we never detected HIV in living and mobile spermatozoa although we sometime found the virus in seminal liquid and in nuclear fractions. Up to date, frozen sperms free of HIV particles from 11 HIV-positive male partners were used to inseminate safely and with success the 11 HIV-negative female partners (no female partner as well as babies contamination). In conclusion, our procedure of HIV-free spermatozoa screening allowed discordant couples to have HIV-negative descendants. In contrast to the natural coïtus in ovulated period or to insemination with washed spermatozoa that are really "Russian roulette", our procedure ensure a total security of no contamination for the pregnant mother.