Plasma levels of plasminogen activator inhibitor type 1, beta- thromboglobulin, and fibrinopeptide A before, during, and after treatment of acute myocardial infarction with alteplase

Plasma levels of plasminogen activator inhibitor type-1 (PAI-1), beta- thromboglobulin (beta TG), and fibrinopeptide A (FPA) were followed over 24 hours in 30 patients treated with alteplase for acute myocardial infarction. Samples were taken at baseline (T Oh), after 90 minutes (under alteplase, no heparin, T 1.5h), after 120 minutes (under alteplase and heparin, T 2h), 30 minutes after thrombolytic therapy (T 3.5h), as well as 12 hours (T 12h) and 24 hours (T 24h) after baseline. PAI-1 antigen levels (55 +/- 9 ng/mL at T Oh, mean +/- SEM) decreased to 35 +/- 5 (T 1.5h) and 40 +/- 6 (T 2h) ng/mL under alteplase, before increasing to 84 +/- 22 (T 3.5h), 130 +/- 30 (T 12h), and 64 +/- 7 (T 24h) ng/mL after therapy, P less than .001. A high baseline PAI-1 activity (18 +/- 3 ng/mL) decreased to 2.0 +/- 0.4 (T 1.5h) and 1.7 +/- 0.2 (T 2h) under alteplase and increased to 32 +/- 5 (T 12h) and 19 +/- 3 (T 24h) ng/mL after therapy (P less than .0001). beta TG levels (339 +/- 105 ng/mL at T Oh) decreased to 203 +/- 48 (T 2h), 154 +/- 51 (T 3.5h), 187 +/- 40 (T 12h), and 142 +/- 32 (T 24h) ng/mL under heparin (P less than .01). FPA levels (34 +/- 9 ng/mL at T Oh) increased to 85 +/- 15 ng/mL under alteplase alone (T 1.5h) and normalized under heparin (11 +/- 4, 6 +/- 2, 4 +/- 2, and 3 +/- 1 ng/mL at T 2h, T 3.5h, T 12h, and T 24h, respectively). A high level of FPA at T 3.5h correlated with reocclusion (33 +/- 12 ng/mL, n = 4 v 2.9 +/- 0.5 ng/mL, n = 21, P less than .005). We conclude that plasma levels of PAI- 1 antigen as well as activity markedly increase after alteplase therapy of acute myocardial infarction. The high activity of PAI-1 and decreasing beta TG levels suggest that platelets do not contribute significantly to this phenomenon. The marked increase of FPA levels under recombinant tissue-type plasminogen activator alone and its normalization under heparin emphasize the important role of concomitant anticoagulation in controlling further intravasal fibrin generation under alteplase.     

Outcome of childhood acute lymphoblastic leukaemia in resource-poor countries

The causes of treatment failure in childhood acute lymphoblastic leukaemia are thought to differ between resource-rich and resource-poor countries. We assessed the records of 168 patients treated for this disease in Honduras. Abandonment of treatment (n=38), the main cause of failure, was associated with prolonged travel time to the treatment facility (2-5 h: hazard ratio 3.1, 95% CI 1.2-8.1 vs >5 h: 3.7, 1.3-10.9) and age younger than 4.5 years (2.6, 1.1-6.3). 35 patients died of treatment-related effects. Outcome could be substantially improved by interventions that help to prevent abandonment of therapy (such as funding for transport, satellite clinics, and support groups), and by prompt treatment of infection.     

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.     

Supratentorial Intraparenchymal Haemorrhages during Spine Surgery

Intracranial haemorrhages are rare but potentially life-threatening complications of spine surgery. Most reported cases involved subdural or cerebellar haemorrhages; supratentorial parenchymal bleeding is very uncommon. We report a 28-year-old woman who underwent resection of a thoracic Ewing''s sarcoma, and developed fatal haemorrhages around her cerebral metastases during surgery. The clinical presentations, possible pathogenesis and potential preventive measures are discussed. Patients with disseminated metastases within the neural axis are at risks of intracranial complications during spine surgery. The presence of intracranial mass lesions should be considered as a relative contraindication to intradural spine surgery.     

Randomized clinical trial of class II restoration in permanent teeth comparing ART with composite resin after 12 months

Clinical Oral Investigations - This study evaluated the effectiveness of class II restorations, in permanent teeth, through the ART technique in comparison to composite resin. Participants (154),...     

Identification and characterization of the constituent human serum antibodies elicited by vaccination

Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT⁺ serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K_d ∼ 10⁻⁸–10⁻¹⁰ M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3⁻CD14⁻CD19⁺CD27⁺⁺CD38⁺⁺CD20⁻TT⁺) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K_d). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.Most approved vaccines confer protection against infectious diseases by the induction of long-lived plasma cells (LLPCs), which secrete antibodies that serve to neutralize and opsonize the pathogen for many years or decades (1–3). Additionally, the generation of memory B cells (mBCs) provides both a mechanism for the rapid synthesis of affinity matured, antigen-specific antibodies following rechallenge and a means to diversify the humoral immune response to confer protection against rapidly evolving viruses or bacteria (4). Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6–7 d and subsequently rapidly decline to nearly undetectable levels (6, 7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).The establishment of serological memory following either primary or booster vaccination is not understood well (10–14). Even though antibody production is the most critical effector function of B-cell immunity, and antigen-specific antibodies in the serum play a key role in protection against pathogen challenge, technical difficulties have precluded direct determination of the identities of the mAbs that comprise the serum antibody response to vaccination. However, recent studies showing that flu vaccination elicits not only neutralizing antibodies but also antibodies that enhance infection by different flu strains (15) underscore the pressing need to develop approaches for delineating the sequences and functionalities of the serum antibodies elicited by vaccination (16).Single-cell cloning has been used to identify neutralizing antibodies encoded by mBCs or plasmablasts in peripheral blood (17). However, although extremely useful for understanding of the structural mechanisms that can lead to the blockade of pathogen infection, the interrogation of single peripheral B cells alone cannot provide information on whether antibodies encoded by single B cells are also produced as secreted IgGs from BM LLPCs, and hence whether they contribute to the serological memory induced by vaccination. A detailed understanding of the diversity of serum antibodies elicited by vaccination, their functionality (e.g., antigen affinity, epitope specificity), and their relative concentrations in the blood can provide key insights toward vaccine evaluation and development.Here, we deployed high-resolution liquid chromatography (LC) tandem MS (MS/MS) (18–20) for the molecular-level analysis of the serum IgG repertoire, combined with deep sequencing of the V gene repertoire of peripheral B lymphocyte subsets (20) and subsequent expression and characterization of representative serum antibodies, to map the dynamics of the human humoral response to vaccination in unprecedented detail. We elected to analyze the response to booster immunization of the TT vaccine because (i) it elicits a highly effective neutralizing response that is protective toward Clostridium tetani challenge; (ii) the vaccine is highly efficacious, and as a result, no deaths from tetanus intoxication have been reported in the United States for individuals who have completed at least primary immunization (21); (iii) TT has been used as a model for analyzing B-cell development following vaccination in humans (6, 22, 23); and (iv) although early serological and mAb studies had pointed to the C-terminal fragment of the toxin heavy chain [recombinant TT fragment C (rTT.C)] as the target for antibody-mediated protection (24), the precise mechanism by which antibodies elicited by the vaccine mediate neutralization has remained unclear.We show that the anti-TT serum IgG repertoire at steady state is composed of a limited number of antibody clonotypes (∼80–100) displaying uniformly high antigen affinity (low nanomolar or subnanomolar), that most of the serum repertoire postboost comprises preexisting (i.e., prevaccination) serum antibody clonotypes, and that there is only partial overlap between the peak-wave plasmablast V gene repertoire and the TT⁺ serum IgG repertoire at steady state after vaccination. We identified several serum monoclonal IgGs that bind to rTT.C, and epitope mapping revealed that all rTT.C-specific antibodies tested bind to an immunodominant linear epitope at the ganglioside-binding site of the toxin that is used for cell entry. Computational antibody docking substantiated that binding of these antibodies to the toxin blocks access to the ganglioside ligand, thus providing a possible mechanistic explanation for how the TT vaccine confers protection. These results highlight the importance of understanding the composition and dynamics of the serum antibody repertoire, together with the V gene repertoire in peripheral B lymphocytes, for the molecular understanding of vaccine function.