Lymphocytes with antigen receptors in drug allergy

The data on the possibility of using the rosette-formation tests for the diagnosis of drug allergy are presented. Tests based on changes in the levels of activated T- and B-lymphocytes after their incubation with allergenic drugs have proved to be low informative. The test found to be highly informative is the antigen-specific rosette-formation test based on the detection of lymphocytes, capable of binding allogeneic erythrocytes loaded with antibiotics causing allergy in patients, in the peripheral blood. This test may be of importance not only in diagnosis, but also for prognosis, as it permits the detection of sensitization to a drug before the clinical manifestations of allergy.     

Allergic alteration of leukocytes in patients with drug intolerance

Increased sensitivity to benzylpenicillin, ampicillin, claforan, cefzol, novocain and lidocaine was determined by means of the leukocyte allergic alteration (LAA) test in 599 patients with intolerance reactions to these preparations in their medical history. In most of these patients the test results exceeded 30% and reached 98%, their average values exceeding those in the control group no subjects with no drug intolerance. The LAA test ensured a high level (83%) of the detection of drug intolerance.     

Rapid, fluorescence-based assay for microtiter plates to test drug influences on neutrophil transmigration through endothelial cell monolayers

Investigation of drug interactions between blood cells and endothelium is of high interest. The current study describes the development of a rapid fluorescence-based leukocyte transmigration system through endothelial cell monolayers for investigation of drug influences. To test the new assay, endothelial cells were cultured on microporous filters, pore size 3.0 microm, in 96-well-plates. Freshly isolated neutrophils were seeded on endothelial cell monolayers and transmigrated cells were measured after incubation for three hours. Migration of non-stimulated neutrophils through non-stimulated endothelial cell monolayer was used as control and set as 100%. The influence of the non-steroidal anti-inflammatory drug diclofenac was investigated. Assay precision tests were done using intraassay (within-day variability) and interassay (day-to-day variability) controls. Transmigration rate was decreased to 53 +/- 6.8% SD (diclofenac 0.7 microg/mL). Different concentrations showed a dose dependent effect (0.07 microg/mL: 97 +/- 9.5%, 7 microg/mL: 37 +/- 4.7%). Analysis of assay accuracy of the new 96-well-sized transmigration assay showed reliable results (coefficient of variation: intraassay 8.2 %; interassay 11.8%). In conclusion, this new, rapid, and sample saving 96-well-microtiter transmigration assay allows examination of drug influence on neutrophil migration through endothelial cell monolayers. Moreover, this assay can also be used for other cell-cell interactions.     

Nonspecific allergy indices in oral sensitization by chicken egg white

Some indices of nonspecific reactivity: the number of cyclic immunological complexes, titer of antibodies, activity of alkaline and acid phosphatases, alanine aminotranspherase, mitochondrial liver H(+)-ATPase, content of glutathione ceruloplasmin transferrin are stated to change in the model of food anaphylaxis in 37 guinea pigs, 26 rats and 12 rabbits orally sensitized with hen egg protein and they are estimated prior to and after the allergy development.     

The role of oral glucose intolerance test in reducing pregnancy complications

The influence of glucose monitoring during pregnancy on newborn body weight, and complications during pregnancy and labor was assessed. We performed a retrospective analysis of macrosomal children, fetal growth, caesarean sections, malformations, still-births and the number of oral glucose tolerance test (OGTT) carried out in a five-year period. The proportion of women participating in OGTT tests increased from 20% to 40% (p<0.05) between 2000 and 2004. Gestation diabetes mellitus (GDM) proportions among pregnant women seen at the Department of Obstetrics and Gynecology at Slavonski Brod General Hospital, Croatia increased from 1% to 6.7% (p < 0. 05) during the observed period. Proportion of births identified as macrosomal decreased from 13.3% to 12.2% (p<0.05). Additionally, infant mortality and still-births along with other fetal and maternal complications declined during the same period. These results suggest that regular measurements of glucose tolerance during pregnancy may prevent preterm birth, decrease the proportion of macrosomal newborns, lower mortality and decrease fetal and maternal complication incidence during pregnancy and delivery.     

In vitro diagnosis of penicillin and streptomycin allergy. The specific leukocyte alteration reaction using luminescence microscopy]

The method of fluorescent microscopy was used for studying the diagnostic value of the reaction of the leucocyte specific alteration (LSA) in patients with different syndromes of hypersensitivity, allergy in the anamnesis and without hypersensitivity to penicillin and streptomycin. It was found that only markedly positive results of the LSA reaction (independent of the sensibilization type) were of diagnostic value, the results of the reaction being stated in half of the patient with hypersensitivity in the anamnesis and in 3/5 of the patients with allergy. Simultaneous use of other tube immunological or skin tests was recommended for the other patients with lower levels of the positive results of the LSA reaction with a purpose of etiological diagnostics or revealing latent sensibilization before treatment with the antibiotics. The LSA reaction is recommended for practical use in complex with other methods of allergological examination.     

Novel antibody assessment method for microbial compositional alteration in the oral cavity

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.     

In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.     

Cutaneous insulin allergy responsive to oral desensitisation and aspirin.

A diabetic man with no previous history of allergy began to suffer itchy, painful swelling at the sites of injection after three months'' treatment with bovine insulin. Insulin specific IgE concentrations (1.2-2.0 U/ml) were higher than in non-allergic diabetics (mean 0.4 (SD 0.06) U/ml) but lower than in most other patients allergic to insulin (1.0-19.0 U/ml). Standard approaches failed to overcome the allergic reaction, and four separate attempts at desensitisation were unsuccessful. The patient was then given oral insulin 800 U thrice daily together with enteric coated aspirin 1300 mg thrice daily for one week, and subsequent desensitisation with neutral insulin injection was carried out successfully. On stopping the aspirin the original reactions returned, and aspirin was therefore reinstituted as a permanent part of treatment. Whatever the mechanism in this patient, oral desensitisation and aspirin provided a simple method for treating a difficult condition.     

The neutrophil in vascular inflammation

Here we focus on how neutrophils have a key regulatory role in vascular inflammation. Recent studies using advanced imaging techniques have yielded new insights into the mechanisms by which neutrophils contribute to defense against bacterial infections and also against sterile injury. In these settings, neutrophils are recruited by various mechanisms depending on the situation. We also describe how these processes may be disrupted in systemic infections, with a particular emphasis on mouse models of sepsis. Neutrophils are often immobilized in the lungs and liver during systemic infections, and this immobilization may be a mechanism through which bacteria can evade the innate immune response or allow neutrophils to form neutrophil extracellular traps that trap and kill bacteria in blood. The platelet is also an important player in sepsis, and we describe how it collaborates with neutrophils in the formation of neutrophil extracellular traps.