Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma.

Mutations in the K-ras oncogene and in the p53 tumor suppressor gene are commonly identified in sporadic cases of pancreatic adenocarcinoma. Although these genes might serve as useful markers for early diagnosis of pancreatic carcinoma in patients at risk for the development of this disease, familial pancreatic carcinomas have not been studied for these mutations. We recently had the opportunity to examine a pancreas prophylactically removed from a patient with a strong family history of pancreatic carcinoma. This gave us the unique opportunity to study the early events in the development of familial adenocarcinoma of the pancreas. Histopathological examination of the pancreas revealed multifocal papillary and nonpapillary mucinous duct hyperplasia. Seven of these foci were microdissected and analyzed for K-ras and p53 mutations. The K-ras mutations were detected by combined mutant-enriched polymerase chain reaction-restriction fragment length polymorphism analysis and characterized further by allele-specific oligonucleotide hybridization. Five of the seven duct lesions harbored activating point mutations in codon 12 of K-ras; a G to A transition was found in four and a G to C transversion in one. In contrast, these lesions did not harbor detectable p53 mutations as determined by denaturing gradient gel electrophoresis of exons 5 to 8, nor was there overexpression of the p53 protein as determined by immunohistochemistry. These findings suggest that mutations in K-ras represent an early event in the pathogenesis of pancreatic carcinoma. In addition, monitoring of patients with a strong family history of pancreatic carcinoma for K-ras mutations may identify patients at risk for the development of invasive carcinoma.     

Use of various genetic markers in differentiation of Mycobacterium bovis strains from animals and humans and for studying epidemiology of bovine tuberculosis.

One hundred fifty-three Mycobacterium bovis strains from cattle, various animal species from zoos and wild parks, and humans were analyzed for three different genetic markers for use in the epidemiology of bovine tuberculosis. M. bovis strains isolated from cattle were found to carry a single IS6110 element, whereas the majority of strains from other animals such as antelopes, monkeys, and seals harbored multiple IS6110 elements, suggesting that the reservoirs in cattle and wild animals are separated. Because the single IS6110 element in cattle strains is located at the same chromosomal position, strain differentiation by insertion sequence fingerprinting was hampered. Therefore, we investigated the usefulness of the direct repeat and polymorphic GC-rich repeat elements for strain differentiation. Both markers allowed sufficient strain discrimination for epidemiological purposes. Evidence is presented that in Argentina, most human M. bovis infections are due to transmission from cattle, whereas M. bovis infections among humans in the Netherlands are mainly contracted from animals other than cattle. Various outbreaks of M. bovis among animals and humans are described, including a small one which likely involved transmission from human to human.     

Endogenous tumor necrosis factor alpha is required for enhanced antimicrobial activity against Toxoplasma gondii and Listeria monocytogenes in recombinant gamma interferon-treated mice.

In vitro studies have shown that macrophages stimulated with recombinant gamma interferon (rIFN-gamma) produce tumor necrosis factor alpha (TNF-alpha), which in an autocrine fashion activates these cells. The aim of the present study was to determine whether endogenously formed TNF-alpha also is required for rIFN-gamma-induced macrophage activation and enhanced antimicrobial activity in vivo. After an intraperitoneal injection of rIFN-gamma into CBA/J mice, their peritoneal macrophages released enhanced amounts of NO2- and inhibited the intracellular proliferation of Toxoplasma gondii. Injection of neutralizing antibodies against TNF-alpha simultaneously with the rIFN-gamma completely inhibited both the release of NO2- by macrophages and their toxoplasmastatic activity. Similar results were observed after intraperitoneal injection of a competitive inhibitor of L-arginine, NG-monomethyl-L-arginine, together with rIFN-gamma, demonstrating that in vivo L-arginine-derived reactive nitrogen intermediates are essential for the induction of toxoplasmastatic activity. Intravenous injection of rIFN-gamma inhibited the growth of Listeria monocytogenes in the livers and spleens of mice; this effect was abrogated by antibodies against TNF-alpha. Intravenous injection of a large dose of rTNF-alpha resulted in a decrease in the number of bacteria in the liver and spleen, but an injection of rIFN-gamma and rTNF-alpha did not result in enhanced inhibition of the proliferation of L. monocytogenes. Together, the results of the present study are the first to demonstrate that endogenous TNF-alpha is required in vivo for the expression of macrophage activation with respect to the release of reactive nitrogen intermediates and toxoplasmastatic activity and for enhanced listericidal activity in the livers and spleens of mice stimulated with rIFN-gamma.     

Urogenital Chlamydia trachomatis serovars in men and women with a symptomatic or asymptomatic infection: an association with clinical manifestations?

To determine whether certain Chlamydia trachomatis serovars are preferentially associated with a symptomatic or an asymptomatic course of infection, C. trachomatis serovar distributions were analyzed in symptomatically and asymptomatically infected persons. Furthermore, a possible association between C. trachomatis serovars and specific clinical symptoms was investigated. C. trachomatis-positive urine specimens from 219 asymptomatically infected men and women were obtained from population-based screening programs in Amsterdam. Two hundred twenty-one C. trachomatis-positive cervical and urethral swabs from symptomatically and asymptomatically infected men and women were obtained from several hospital-based departments. Serovars were determined using PCR-based genotyping, i.e., restriction fragment length polymorphism analysis of the nested-PCR-amplified omp1 gene. The most prevalent C. trachomatis serovars, D, E, and F, showed no association with either a symptomatic or asymptomatic course of infection. The most prominent differences found were (i) the association of serovar Ga with symptoms in men (P = 0.0027), specifically, dysuria (P < 0.0001), and (ii) detection of serovar Ia more often in asymptomatically infected people (men and women) (P = 0.035). Furthermore, in women, serovar K was associated with vaginal discharge (P = 0.002) and serovar variants were found only in women (P = 0.045).     

Testing bronchial hyper-responsiveness: provocation or peak expiratory flow variability?

BACKGROUND: Assessing bronchial hyper-responsiveness (BHR) is a main diagnostic criterion of asthma. Provocation testing is not readily available in general practice, but peak expiratory flow (PEF) is. Several guidelines promote the use of PEF variability as a diagnostic tool for BHR. This study tested the agreement between histamine challenge testing and PEF variability, and the consequences for diagnosing asthma. AIM: To investigate the possibility of assessing BHR by PEF variability, using a histamine provocation test as a reference. METHOD: Subjects with signs of symptoms indicating asthma (persistent or recurrent respiratory symptoms or signs of reversible bronchial obstruction) (n = 323) were studied. They had been identified in a population screening for asthma. A histamine provocation test and PEF variability were assessed over a three-week period. Asthma was defined as signs or symptoms together with a reversible airflow obstruction or BHR to the histamine challenge test. BHR was defined as a PC20 histamine of < or = 8 mg/ml or a PEF variability of > or = 15%. Overall correlation between PC20 and PEF variability was calculated using Spearman's rho. Furthermore, a decision tree was constructed to clarify the role of BHR in diagnosing asthma. RESULTS: Thirty-two patients had a reversibility in forced expiratory volume in 1 second (FEV1) of > or = 9% predicted, 131 patients showed a PC20 of < or = 8 and 11 patients had a PEF variability of > or = 15%. Overall correlation was poor at only -0.27 (P < 0.0001). One hundred and fourteen of the 131 patients diagnosed as having asthma when the histamine challenge test was used were not diagnosed by PEF variability. CONCLUSION: PEF variability cannot replace bronchial provocation testing in assessing BHR. This indicates that PEF variability and bronchial provocation do not measure the same aspects of BHR. If BHR testing is required in diagnosing asthma, a bronchial provocation test has to be used in general practice as well.     

Parameters of T cell mediated immunity to commensal micro-organisms in patients with chronic purulent rhinosinusitis: a comparison between delayed type hypersensitivity skin test, lymphocyte transformation test and macrophage migration inhibition factor assay.

In 75 patients with unexplained chronic purulent rhinosinusitis T cell mediated immunity to three micro-organisms frequently colonizing the human upper respiratory tract, viz. Haemophilus influenzae, streptococci and Candida albicans, was assessed. Delayed type hypersensitivity (DTH) skin test reactivity was measured in vivo, whereas the blastogenic responsiveness (lymphocyte transformation test; LTT) and lymphokine production (e.g. migration inhibition factor; MIF) of the lymphocytes upon antigen stimulation were measured in vitro. MIF was assayed with a recently developed test system using the human monocytoid cell-line U937 as indicator cells in agarose microdroplets. Two-thirds of the 75 patients tested showed a defective DTH response to one or more of the microbial antigens; this contrasted to the findings in 25 healthy subjects, of whom over 90% showed a positive DTH reaction to any of the three antigens. PHA skin tests were entirely normal in both patients and healthy controls. Microbial antigen-specific LTT responses fluctuated considerably in time from strongly positive to negative and vice versa in healthy individuals as well as in patients. In general however, blastogenic responses in patients were comparable to or even higher than those of healthy persons. In the MIF assay, lymphocytes of all healthy individuals tested showed production of MIF upon stimulation with all three antigens; this again contrasted to two-thirds of the patients, whose lymphocytes showed a defective MIF production. Fluctuations of MIF-production in time could not be established and a very good correlation existed between the data obtained in the MIF assay and those of the DTH skin tests. These results indicate that apart from skin testing, the MIF assay seems to be the most suitable parameter to assess defects in T cell reactivity towards microbial antigens. These defects exist in two-thirds of our patients suffering from chronic purulent rhinosinusitis.     

Aggregation of human polymorphonuclear leucocytes during phagocytosis of bacteria.

The process of aggregation of human polymorphonuclear leucocytes (PMN) during the uptake of bacteria was studied. Radiolabelled S. aureus were opsonized in different sera, washed, resuspended in buffer and added to the PMN. Uptake of the bacteria and aggregation of the PMN were measured simultaneously. Maximal aggregation occurred within 6 min, when 5 X 10(6) PMN had phagocytosed 2.5 X 10(8) S. aureus. Also the effects of serum concentrations and different sera for opsonization of the bacteria on PMN aggregation were studied. Despite normal uptake, aggregation of PMN was low when bacteria were opsonized in complement-deficient sera. Furthermore when PMN were treated with pronase to inactivate complement receptors on the cell surface of the PMN, and bacteria preopsonized in immune serum were added, no change in uptake occurred, although the degree of aggregation halved compared to control PMN. So, interaction between the bacteria and the complement receptor of the PMN cell membrane is needed for triggering the process of aggregation. By using dansylcadaverin and diphenylamine to modulate lysosomal enzyme release, azide or PMN from a chronic granulomatous disease patient to study the effect of the formation of oxygen species, and theophylline, DB-cAMP or 8 Br-cAMP to increase cAMP levels, it was concluded that aggregation of PMN during phagocytosis was not dependent on oxygen metabolism, degranulation or cAMP levels of PMN.     

Adaptive filtering of canine electrogastrographic signals. Part 2: filter performance

The modified adaptive filter method described in Part 1 was applied to 16 stretches of (cutaneous) electrogastrographic signal of 17·07 min duration. A signal-to-noise ratio improvement of about 8 dB was achieved. The most characteristic feature of the filter method appeared to be that wave-form and phase of the gastric component of the electrogastrographic signal are preserved. It is concluded that the use of the modified adaptive filter forms a valuable tool in the study of the electrogastrographic signal.     

X-linked mixed deafness (DFN3): cloning and characterization of the critical region allows the identification of novel microdeletions

We have found that the microsatellite marker AFM207zg5 (DXS995)maps to all previously described deletions which are associatedwith X-linked mixed deafness (DFN3) with or without choroideremiaand mental retardation. Employing this marker and pHU16 (DXS26)we have identified two partially overlapping yeast artificialchromosome clones which were used to construct a complete 850kb cosmid contig. Cosmids from this contig have been testedby Southern blot analysis on DNA from 16 unrelated males withX-linked deafness. Two novel microdeletions were detected inpatients which exhibit the characteristic DFN3 phenotype. Bothdeletions are completely contained within one of the known DFN3-deletions,but one of them does not overlap with two previously describeddeletions in patients with contiguous gene syndromes consistingof DFN3, chorolderemia, and mental retardation. Assuming thatonly a single gene is involved, this suggests that the DFN3gene spans a chromosomal region of at least 400 kb.