Immunoglobulin (Ig)M antibodies to proteinase 3 in granulomatosis with polyangiitis and microscopic polyangiitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) appear to play an important role in the pathogenesis of ANCA-associated vasculitis (AAV). However, ANCA alone are not sufficient to generate disease, and some evidence suggests that infectious triggers may serve as inciting events for AAV disease activity. Antibodies of the immunoglobulin (Ig)M isotype often serve as markers of recent infection, and IgM ANCA have been identified previously in patients with AAV, although the frequency and clinical relevance of IgM ANCA is not well established. We sought to characterize IgM ANCA more clearly by creating a novel enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to proteinase 3 [IgM proteinase 3 (PR3)-ANCA], which we applied to two large, clinically well-characterized trial cohorts of patients with granulomatosis with polyangiitis and microscopic polyangiitis. In the first cohort, IgM PR3-ANCA occurred with a frequency of 15·0%, and were associated with a higher degree of disease severity and a trend towards a higher rate of alveolar haemorrhage (29·6 versus 15·7%, P = 0·10). Analysis of follow-up samples in this cohort showed that the presence of IgM PR3-ANCA was transient, but could recur. In the second cohort, IgM PR3-ANCA occurred with a frequency of 41·1%, and were also associated with a higher degree of disease severity. A higher rate of alveolar haemorrhage was observed among those with IgM PR3-ANCA (45·3 versus 15·8%; P < 0·001). The association of transient IgM PR3-ANCA with an acute respiratory manifestation of AAV suggests a possible link between an infectious trigger and AAV disease activity.

Development and Optimization of a Selective Whole-Genome Amplification To Study Plasmodium ovale Spp.

Since 2010, the human-infecting malaria parasite Plasmodium ovale spp. has been divided into two genetically distinct species, P. ovale wallikeri and P. ovale curtisi. In recent years, application of whole-genome sequencing (WGS) to P. ovale spp. allowed to get a better understanding of its evolutionary history and discover some specific genetic patterns. Nevertheless, WGS data from P. ovale spp. are still scarce due to several drawbacks, including a high level of human DNA contamination in blood samples, infections with commonly low parasite density, and the lack of robust in vitro culture. Here, we developed two selective whole-genome amplification (sWGA) protocols that were tested on six P. ovale wallikeri and five P. ovale curtisi mono-infection clinical samples. Blood leukodepletion by a cellulose-based filtration was used as the gold standard for intraspecies comparative genomics with sWGA. We also demonstrated the importance of genomic DNA preincubation with the endonuclease McrBC to optimize P. ovale spp. sWGA. We obtained high-quality WGS data with more than 80% of the genome covered by ≥5 reads for each sample and identified more than 5,000 unique single-nucleotide polymorphisms (SNPs) per species. We also identified some amino acid changes in pocdhfr and powdhfr for which similar mutations in P. falciparum and P. vivax are associated with pyrimethamine or cycloguanil resistance. In conclusion, we developed two sWGA protocols for P. ovale spp. WGS that will help to design much-needed large-scale P. ovale spp. population studies. IMPORTANCE Plasmodium ovale spp. has the ability to cause relapse, defined as recurring asexual parasitemia originating from liver-dormant forms. Whole-genome sequencing (WGS) data are of importance to identify putative molecular markers associated with relapse or other virulence mechanisms. Due to low parasitemia encountered in P. ovale spp. infections and no in vitro culture available, WGS of P. ovale spp. is challenging. Blood leukodepletion by filtration has been used, but no technique exists yet to increase the quantity of parasite DNA over human DNA when starting from genomic DNA extracted from whole blood. Here, we demonstrated that selective whole-genome amplification (sWGA) is an easy-to-use protocol to obtain high-quality WGS data for both P. ovale spp. species from unprocessed blood samples. The new method will facilitate P. ovale spp. population genomic studies.

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface.

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze-fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed.

Frequency of visualization of presumed celiac ganglia by endoscopic ultrasound.

BACKGROUND AND STUDY AIMS: Celiac ganglia can be visualized by endoscopic ultrasound (EUS). It is unknown how often ganglia are visualized during EUS, and what clinical factors are associated with ganglion visualization. The aim of this study was to prospectively evaluate the frequency of visualization of presumed celiac ganglia by EUS and to identify factors that predict their visualization. PATIENTS AND METHODS: Clinical, demographic, EUS, and cytologic data were collected prospectively from 200 unselected patients who were undergoing EUS in a tertiary referral centre. When presumed celiac ganglia were visualized, their size, number, location, and echo features were noted. When presumed ganglia were aspirated, the results of cytology were recorded. RESULTS: The most common indication for EUS was investigation of a pancreatic mass or cyst (25 %). Presumed celiac ganglia were identified in 81 % of patients overall. Logistic regression analysis determined that female sex and having no prior history of gastrointestinal surgery were independently associated with ganglion visualization. Among patients whose ganglia were visualized, more ganglia were seen per patient with linear echo endoscopes (2, range 0 - 5) than with radial echo endoscopes (1, range 0 - 4) ( P = 0.001). Presumed celiac ganglia were aspirated in 10 patients; and cytologic examination revealed neural ganglia in all of these. CONCLUSIONS: Celiac ganglia can be visualized by EUS in most patients who undergo upper gastrointestinal EUS examinations, and are best seen with linear-array echo endoscopes. Ganglia can usually be differentiated from lymph nodes on the basis of their endosonographic appearance.

Preferential expression of domain cassettes 4, 8 and 13 of Plasmodium falciparum erythrocyte membrane protein 1 in severe malaria imported in France.

OBJECTIVES: Severe Plasmodium falciparum malaria (SM) involves cytoadhesion of parasitized red blood cells, mediated by P. falciparum erythrocyte membrane protein 1, which is encoded by var genes. Expression of var gene group A and B or encoding domain cassettes DC4, DC5, DC8 and DC13 has been implicated in SM in African children, but no data exist in the context of imported malaria. The aim of this study was to investigate var gene expression linked to clinical presentation and host factors in SM imported into France. METHODS: Expression level of var gene groups A, B, C, var1, var2csa, var3 and var genes encoding DC4, DC5, DC8 and DC13 was measured by quantitative RT-PCR and expressed in transcript units. Seventy SM and 48 uncomplicated malaria (UM) P. falciparum cases were analysed according to disease severity, epidemiological characteristics (migrants or travellers) and anti-P. falciparum antibodies. Cluster analysis was performed to identify gene expression profiles. RESULTS: Var1 and B/C expression were higher in UM than SM (0.66 (0-1.1) and 1.88 (1.3-2.4); p <0.04, respectively). Group C expression differed between migrants and travellers (0.21 (0-0.75) versus 0 (0-0); p 0.002). Group A differed in naive and pre-exposed patients (1.1 (0.7-1.5) versus 0.4 (0-1.1); p 0.01). Population clusters revealed increased expression from group A and B var genes, and DC4, DC8 and DC13 in SM. CONCLUSIONS: These results corroborate the implication of DC4, DC8 and DC13 in severe imported malaria cases as African children, and their expression depends of host factors.

[Antimalarial drug resistance]. / Les résistances aux médicaments antipaludiques.

Drug resistant malaria is mostly due to Plasmodium falciparum, the highly prevalent species in tropical Africa, Amazon, and Southeast Asia. P. falciparum is responsible for severe involvement of fever or anemia causing more than a million deaths per year. Rationale for treatment is becoming weak as multiple drug resistance against well-tolerated drugs develops. P. falciparum drug resistant malaria originates from chromosomal mutations. Analyses using molecular, genetic and biochemical approaches showed that: 1) impaired uptake of chloroquine by the parasite vacuole is a common characteristic of resistant strains, this phenotype correlates with pfmdr1 and pfcrt gene mutations; 2) one S108N to four (N51I, C59R, I164L) point mutations of dihydrofolate reductase, the enzyme target of antifolinics (pyrimethamine and proguanil), give moderate to high level of resistance to these drugs; 3) resistance to sulfonamides and sulfones involves mutations of dihydropteroate synthase (A437G, K540E), their enzyme target, impairing their capacity to potentiate antifolinic drugs; 4) resistance to atovaquone plus proguanil involves one single mutation on atovaquone target, cytochrome b (Y268S, C or N); 5) resistance to mefloquine is thought to be linked to the over expression of pfmdr1, a pump expelling toxic waste from eukaryotic cells. P. falciparum resistance levels may differ according to places and time, depending on malaria transmission and drug pressure. Coupling in vivo to in vitro tests, and using molecular tests is essential for the surveillance of replacement drugs. Low cost biochemical tools are urgently needed for a prospective monitoring of resistance.

Structure-function analysis of a double-mutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis.

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype.

Optimising outcomes in acute pancreatitis.

Acute pancreatitis is a common cause for presentation to emergency departments. Common causes in Western societies include biliary pancreatitis and alcohol (the latter in the setting of chronic pancreatitis). Acute pancreatitis also follows endoscopic retrograde pancreatography in 5 to 10% of patients, a group that could potentially benefit from prophylactic treatment. Although episodes of pancreatitis usually run a relatively benign course, up to 20% of patients have more severe disease, and this group has significant morbidity and mortality. Therefore, attempts have been made to identify, at or soon after presentation, those patients likely to have a poor outcome and to channel resources to this group. The mainstay of treatment is aggressive support and monitoring of those patients likely to have a poor outcome. Pharmacotherapy for acute pancreatitis (both prophylactic and in the acute setting) has been generally disappointing. Efforts initially focused on protease inhibitors, of which gabexate shows some promise as a prophylactic agent. Agents that suppress pancreatic secretion have produced disappointing results in human studies. Infection of pancreatic necrosis is associated with high mortality and requires surgical intervention. In view of the seriousness of infected necrosis, the use of prophylactic antibacterials such as carbapenems and quinolones has been advocated in the setting of pancreatic necrosis. Similarly, data are accumulating to support the use of prophylactic antifungal therapy. Recently, it has become apparent that the intense inflammatory response associated with acute pancreatitis is responsible for much of the local and systemic damage. With this realisation, future efforts in pharmacotherapy are likely to focus on suppression or antagonism of pro-inflammatory cytokines and other inflammatory mediators. Similarly, animal studies have demonstrated the importance of oxidative stress in acute pancreatitis, although to date there is a paucity of information regarding the efficacy of antioxidants. Although the clinical course for most patients with acute pancreatitis is mild, severe acute pancreatitis continues to be a clinical challenge, requiring a multidisciplinary approach of physician, intensivist and surgeon.

Influence of hepatic reserve and cause of esophageal varices on survival and rebleeding before and after the introduction of sclerotherapy: a retrospective analysis.

Esophageal variceal sclerotherapy has been enthusiastically accepted as the procedure of choice for patients with variceal hemorrhage. Because the relationships among liver function, different causes of varices, survival, and rebleeding rates have not been well established in sclerotherapy trials, this enthusiasm may be unjustified. We studied these relationships in 80 patients with bleeding esophageal varices who were admitted to hospitals affiliated with our clinic between 1978 and 1980 and who did not receive sclerotherapy and in 162 patients admitted between 1980 and 1982 who received sclerotherapy with ethanolamine oleate. In both groups of patients, survival and bleeding-free intervals were significantly related (P less than 0.005 and P less than 0.01, respectively) to hepatic reserve (Child's class). In addition, patients with nonalcohol-related liver disease and poor hepatic reserve (Child's class C) had reduced survival and bleeding-free intervals compared with patients in class C with alcohol-related liver disease. Similar probabilities of survival and bleeding-free intervals were noted for Child's class subgroups and etiologic subgroups in the sclerotherapy and nonsclerotherapy groups, although a formal comparison was not made because of the retrospective nature of this study. Indications that sclerotherapy increases survival and reduces rebleeding may be due to different distributions of Child's classes and causes of varices within sclerotherapy and nonsclerotherapy groups in published control trials.

Utility of endoscopic ultrasonography in endoscopic drainage of pancreatic pseudocysts in selected patients.

OBJECTIVES: To determine the effect of endoscopic ultrasonography (EUS) on endoscopic drainage of pancreatic pseudocysts and to determine patency with fistula dilation and placement of multiple stents. PATIENTS AND METHODS: Between September 1995 and January 1999, 19 patients underwent endoscopic drainage of pancreatic pseudocysts, 17 of whom were assessed by EUS before drainage. Radial EUS scanning was used to detect an optimal site of apposition of pseudocyst and gut wall, free of intervening vessels. A fistula was created with a fistulatome, followed by balloon dilation of the fistula tract. Patency was maintained with multiple double pigtail stents. The primary goal of this retrospective study was to determine whether EUS affected the practice of endoscopic drainage of pancreatic pseudocysts. RESULTS: In 3 patients, drainage was not attempted based on EUS findings. In the other 13 patients (14 pseudocysts), creation of a fistula was successful on 13 occasions, and no immediate complications occurred. However, 1 patient subsequently developed sepsis that required surgery. All other patients were treated with balloon dilation, multiple stents, and antibiotics, with no septic complications. Of 14 pseudocysts (in 13 patients), 13 (93%) resolved. CONCLUSIONS: Results of EUS may alter management of patients considered for endoscopic drainage of pancreatic pseudocysts. Endoscopic ultrasonography was useful for selecting an optimal and safe drainage site. The combination of balloon dilation, multiple stents, and antibiotics appears to resolve pancreatic pseudocysts without septic complications.

The use of gamma-glutamyl transpeptidase in differentiating liver from bone isoenzymes of alkaline phosphatase.

Sixty-one patients with elevated alkaline phosphatase activity due to liver or bone diseases were studied. An attempt was made to identify the origin of the increased alkaline phosphatase by chemical inhibition, by inactivation by heat and urea, and by electrophoretic separation. The results obtained from these procedures were correlated with the gamma-glutamyl transpeptidase activities performed on each patient. We concluded from this study that gamma-glutamyl transpeptidase determination, together with alkaline phosphatase electrophoretic separations, are useful laboratory procedures for accurately identifying the origin of elevated alkaline phosphatase activity.

Diagnosis of chronic pancreatitis. Is a gold standard necessary?

The question that serves as this article's title is rhetorical. Clinicians have diagnosed and managed chronic pancreatitis without a gold standard for decades and must continue to do so in the foreseeable future. Although clinicians have a much wider array of diagnostic tools available for the diagnosis of chronic pancreatitis, a single readily applied gold standard remains elusive. Diagnostic studies are rarely compared with a true gold standard--histopathology. Furthermore, even if a safe biopsy technique were available, it might fall short of a gold standard, given the patchy nature of early-stage chronic pancreatitis. Indeed, different stages of chronic pancreatitis require not only recognition of the different clinical presentations but also different levels of intensity of diagnostic testing to establish the diagnosis confidently. The diagnosis in most patients with chronic pancreatitis can be made confidently with a good clinical history and a limited number of currently available structural and functional tests. No single diagnostic study, functional or structural, suffices for all patients. It is also axiomatic that patients with intractable abdominal pain in whom early-stage chronic pancreatitis is suspected represent a challenge for clinicians partly because of this lack of a single, dependable gold standard. Perhaps we have reached the point at which further refinements of current tests of structure or function are not beneficial because increased sensitivity is countered by loss of specificity. We suggest that a new approach to developing a gold standard for the diagnosis of chronic pancreatitis is necessary. With advances in the understanding of the mediators of the inflammatory process, it may be possible to devise a test to assess the earliest events in this disease.

Wetting transitions at soft, sliding interfaces.

We observe (by optical interferometry) the contact of a rubber cap squeezing a nonwetting liquid against a plate moving at velocity U. At low velocities, the contact is dry. It becomes partially wet above a threshold velocity V(c1), with two symmetrical dry patches on the rear part. Above a second velocity V(c2), the contact is totally wet. This regime U>V(c2) corresponds to the hydroplaning of a car (decelerating on a wet road). We interpret the transitions at V(c1), V(c2) in terms of a competition between (a) liquid invasion induced by shear (b) spontaneous dewetting of the liquid (between nonwettable surfaces).

[Optimum quinine therapy in falciparum malaria attacks contracted in Africa]. / Du bon usage thérapeutique de la quinine dans l'accès palustre à P. falciparum contracté en Afrique.

A review about the use of quinine for uncomplicated falciparum malaria contracted in Africa is proposed. The dose of 8 mg/kg of quinine base 3 times a day seems to be admitted by all. On the other way, the duration of treatment fluctuates from 2 to 10 days without evidence of difference in efficacy between 3, 5 and 7 days. The pharmacodynamic and pharmacokinetic properties of quinine are reviewed and suggest that 3, 5 and 7 days are efficient at least on, respectively, 1, 2 and 3 parasite cycles and suggest that a five day treatment may be curative for all kind of patients infested with a quinine-sensitive strain of P. falciparum in Africa.

The role of transabdominal ultrasonography, helical computed tomography, and magnetic resonance cholangiopancreatography in diagnosis and management of pancreatic disease.

Many technical advances have offered enhanced capabilities in noninvasive imaging of the pancreas. Although these technical advances are impressive, current studies do not always define clearly the benefits that these advances will confer in patient management. A critical overview of these imaging modalities is offered here, with respect to diagnosis and patient management. Outcomes from various studies are summarized for modalities including transabdominal ultrasound, computed tomography, magnetic resonance imaging with and without pancreatography, and positron emission tomography.

Diabetic diarrhoea and steatorrhoea. A case report and review of the literature.

A case of severe diabetic steatorrhoea is presented in a young Black male with poorly controlled diabetes, symptomatic peripheral neuropathy, evidence of autonomic neuropathy with gastroparesis and raised faecal fat excretion. A pancreatic function test was negative as were tests of small-bowel structure and function. There was resistance to all therapy. The literature on diabetic diarrhoea and steatorrhoea has been reviewed, and it is concluded that they are expressions of the same entity which remains a clinical problem for which there is at present no effective management.