Comparison of LCx with other current viral load assays for detecting and quantifying human immunodeficiency virus type 1 RNA in patients infected with the circulating recombinant form A/G (CRF02)

LCx was compared to other assays in measuring human immunodeficiency virus type 1 (HIV-1) CRF02 viremia. LCx showed significant but low correlation with the other methods. Values of <2.60 log(10) cp/ml were observed in 29.6% of specimens with LCx and in only 14.8% with bDNA and PCR, suggesting suboptimal performance of LCx with CRF02.     

Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis

Autosomal-recessive osteopetrosis is a severe genetic disease caused by osteoclast failure. Approximately 50% of the patients harbor mutations of the ATP6i gene, encoding for the osteoclast-specific a3 subunit of V-ATPase. We found inactivating ATP6i mutations in four patients, and three of these were novel. Patients shared macrocephaly, growth retardation and optic nerve alteration, osteosclerotic and endobone patterns, and high alkaline phosphatase and parathyroid hormone levels. Bone biopsies revealed primary spongiosa lined with active osteoblasts and high numbers of tartrate-resistant acid phosphatase (TRAP)-positive, a3 subunit-negative, morphologically unremarkable osteoclasts, some of which located in shallow Howship lacunae. Scarce hematopoietic cells and abundant fibrous tissue containing TRAP-positive putative osteoclast precursors were noted. In vitro osteoclasts were a3-negative, morphologically normal, with prominent clear zones and actin rings, and TRAP activity more elevated than in control patients. Podosomes, alphaVbeta3 receptor, c-Src, and PYK2 were unremarkable. Consistent with the finding in the bone biopsies, these cells excavated pits faintly stained with toluidine blue, indicating inefficient bone resorption. Bone marrow transplantation was successful in all patients, and posttransplant osteoclasts showed rescue of a3 subunit immunoreactivity.     

Study of the toughening mechanisms in bone and biomimetic hydroxyapatite materials using Raman microprobe spectroscopy

A Raman microprobe spectroscopy characterization of microscopic fracture mechanisms is presented for a natural hydroxyapatite material (cortical bovine femur) and two synthetic hydroxyapatite-based materials with biomimetic structures-a hydroxyapatite skeleton interpenetrated with a metallic (silver) or a polymeric (nylon-6) phase. In both the natural and synthetic materials, a conspicuous amount of toughening arose from a microscopic crack-bridging mechanism operated by elasto-plastic stretching of unbroken second-phase ligaments along the crack wake. This mechanism led to a rising R-curve behavior. An additional micromechanism, responsible for stress relaxation at the crack tip, was recognized in the natural bone material and was partly mimicked in the hydroxyapatite/silver composite. This crack-tip mechanism conspicuously enhanced the cortical bone material resistance to fracture initiation. A piezo-spectroscopic technique, based on a microprobe measurement of 980 cm(-1) Raman line of hydroxyapatite, enabled us to quantitatively assess in situ the microscopic stress fields developed during fracture both at the crack tip and along the crack wake. Using the Raman piezo-spectroscopy technique, toughening mechanisms were assessed quantitatively and rationally related to the macroscopic fracture characteristics of hydroxyapatite-based materials.     

Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (n = 10 samples) or the lack of infected HUVEC with throat washes (n = 3) or amniotic fluid samples (n = 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.     

Interleukin-6 and flow-mediated dilatation as markers of increased vascular inflammation in women receiving hormone therapy

OBJECTIVE: The lack of a beneficial long-term cardiovascular effect of hormone therapy and the early incidence of cardiovascular adverse events observed in recent randomized studies have been related to a heightened inflammatory effect of hormone therapy. DESIGN: We evaluated the effect of different postmenopause therapies on inflammatory markers and endothelial function in 205 postmenopausal women before and after therapy. RESULTS: all postmenopausal women, estrogens alone increased plasma levels of C-reactive protein (CRP) but decreased all other markers of inflammation including interleukin-6 (IL-6) (CRP: +75% +/- 11%, intracellular adhesion molecule: -21% +/- 4%, vascular cell adhesion molecule: -15% +/- 6%, E-selectin: -18% +/- 4%, s-thrombomodulin -10.5% +/- 3.7%, IL-6 -14% +/- 6%; percent changes, P < 0.01 compared with baseline). Raloxifene and tibolone did not significantly affect the overall inflammatory milieu. In a minority of patients, estrogen-progestogen associations and tibolone increased IL-6 levels and induced unfavorable changes on inflammation markers (CRP: +93% +/- 8%, intracellular adhesion molecule: -3% +/- 2%, vascular cell adhesion molecule: -5% +/- 2%, E-selectin: +6% +/- 2%, s-thrombomodulin: +5% +/- 2%, IL-6: +12% +/- 4%; percent changes compared with baseline). Patients with increased IL-6 levels were older and had a longer time since menopause. In all patients except those with increased IL-6 levels, hormone therapy improved endothelial function, whereas tibolone and raloxifene did not significantly change endothelial function compared with baseline. A worsening of endothelial function was detected in patients with increased IL-6 levels during therapy. CONCLUSIONS: Postmenopausal hormone therapy is associated with decreased vascular inflammation; however, in patients with a longer time since menopause, postmenopause hormone therapy may increase inflammation and worsen endothelial function. These unfavorable vascular effects may be detected by an elevation in IL-6 levels and by a lack of improvement in endothelial function.     

Diagnostic performance of aPS/PT antibodies in neuropsychiatric lupus and cardiovascular complications of systemic lupus erythematosus

Abstract

Background: Systemic lupus erythematosus (SLE) is associated with a constellation of complications affecting multiple organs, including neuropsychiatric manifestations (NPSLE) and ischaemic events, leading to increased long-term morbidity. Antiphospholipid antibodies (aPL) are a major determinant of vascular inflammation and thromboembolic risk. The diagnostic role of anti-phosphatidylserine/prothrombin (aPS/PT) antibodies in this setting is incompletely defined.

Aim: To verify whether aPS/PT add to diagnostics and disease stratification in patients with SLE with or without other aPL.

Methods: 131 consecutive patients were studied, including 20 patients with SLE and secondary antiphospholipid syndrome (APS). aPS/PT IgG and IgM were assessed through ELISA and patients were stratified based on the presence of other aPL, on their clinical and laboratory features at time of blood sampling and on their clinical history. Synthetic indices of disease activity, chronic damage and cardiovascular risk were calculated at time of venipuncture.

Results: Fifty-one (38.9%) patients with SLE had aPS/PT and 15 (11.5%) patients had aPS/PT as the only aPL (aPS/PT-only). aPS/PT-only patients had a significantly higher prevalence of NPSLE than quadruple aPL-negative patients (p?=?.007). Patients with aPS/PT were more likely to have a history of ischaemia, thrombocytopenia and Libman–Sacks’ endocarditis. The presence of aPS/PT also associated with previous accrual of at least one damage item (p?=?.043), but had limited predictive values for damage progression in the short term.

Conclusion: aPS/PT antibodies provide non-redundant information that could contribute to risk assessment and stratification of patients with SLE.     

The long pentraxin PTX3 up-regulates tissue factor in activated monocytes: another link between inflammation and clotting activation

Pentraxin-3 (PTX3), an acute-phase protein that belongs to the family of the PTXs, is found elevated in septic shock and increased in patients with acute myocardial infarction. As tissue factor (TF) plays a key role in thrombosis and inflammation associated with atherosclerosis and as we have recently reported that PTX3 increases TF synthesis in endothelial cells, we tested whether PTX3 could modulate TF expression in monocytes. Monocytes from peripheral blood of healthy donors were incubated with highly purified PTX3 with or without lipopolysaccharide (LPS). Cells were then disrupted, and procoagulant activity was assessed by a one-stage clotting time. PTX3 enhanced TF activity and antigen from LPS-stimulated monocytes in a dose-dependent way. The effect was specific, as other PTXs, such as C-reactive protein and serum amyloid P component, were ineffective. Moreover, the increase in activity was specific for LPS, as in the presence of other TF-inducing agents such as interleukin-1beta and tumor necrosis factor alpha, PTX3 was not effective. The increase in TF activity requires mRNA synthesis, as assessed by polymerase chain reaction. The mechanism by which PTX3 modulates TF synthesis resides in an enhanced IkappaB, alpha phosphorylation and degradation and increased migration of the transacting factor c-Rel/p65 into the nucleus, as determined by Western blot and electro-mobility shift assay. These results show that PTX3 is an enhancer of the expression of TF by mononuclear cells. In the area of vascular injury, during the inflammatory response, cell-mediated fibrin deposition takes place. PTX3 increases TF expression, thus potentially playing a role in thrombogenesis and wound healing.     

The advantage of mucosal immunization for polysaccharide-specific memory responses in early life

The aim of vaccination is to rapidly elicit protective immunity and generate memory for sustained protection. We studied the induction and persistence of polysaccharide (PS)-specific memory in neonatal and infant mice primed with pneumococcal conjugate (Pnc1-TT) by assessing the response to native pneumococcal PS (PPS-1), the kinetics of the PPS-1-specific IgG response to a second Pnc1-TT dose and affinity maturation. A subcutaneous (s.c.) Pnc1-TT booster induced a rapid increase in PPS-1-specific IgG, indicating efficient priming for memory by a single dose of Pnc1-TT already at 1 week of age. High levels were maintained for >12 weeks. However, a PPS-1 booster induced no response in neonatal or infant mice. The adjuvant LT-K63 significantly enhanced the IgG response and affinity to Pnc1-TT by both the s.c. and the intranasal (i.n.) route in all age groups. In neonatal and infant mice, PPS-1 and LT-K63 induced a booster response only when given i.n. following either s.c. or i.n. priming with Pnc1-TT and LT-K63. In contrast, PPS-1 with or without LT-K63 administered s.c. compromised the ongoing PPS-1-specific response elicited in neonatal mice by either s.c. or i.n. priming with Pnc1-TT and LT-K63. These results demonstrate the advantage of the mucosal route for elicitation of PS-specific memory responses in early life.     

Enhanced Phagocytosis and Bactericidal Activity of Hepatic Reticuloendothelial System During Endotoxin Tolerance

The effects of tolerance to Escherichia coli endotoxin on the phagocytic and bactericidal activity of the hepatic reticuloendothelial system against viable E. coli were examined using ex vivo perfused rat livers. Livers were isolated from control and endotoxin-tolerant rats and perfused with a medium containing 5% homologous serum from either control or tolerant rats. After the addition of the E. coli (2 × 10⁷ cells per ml) to the perfusate, the hepatic clearance of the bacteria was followed for 30 min. The highest activation of the hepatic reticuloendothelial system was observed when serum from tolerant animals was added to the perfusate. Under these conditions phagocytosis was 47% (12% in controls), and 37 to 38% of the bacteria were killed (5% in controls). This activation was less when livers obtained from tolerant rats were perfused with serum from controls or with saline only. The data suggests that, during endotoxin tolerance, humoral factors play an important role in the activation of the hepatic reticulendothelial system, although a direct stimulation of Kupffer cells also occurs. The enhancement of phagocytosis by tolerant serum did not require the presence of homologous antibodies and involved the activation of the alternative complement pathway, since it was lost after removal of factor B activity. On the other hand, stimulation of intracellular killing required both complement and specific antibodies. The data suggest a role of endotoxin in the activation of humoral and cellular mechanisms involved in the host resistance to gram-negative bacterial infection.