Negative oral provocation test with porcine pancreatic enzyme plus cofactors despite confirmed Alpha-Gal syndrome

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Telediagnosis of oral lesions in primary care: The EstomatoNet Program.

OBJECTIVE: The diagnosis of oral lesions is often challenging for primary healthcare providers, which explains the high number of referrals to specialist care. This favors increases in waiting lines and delays in diagnosis, contributing to high mortality rates from oral cancer. This study aimed to summarize the experience of the EstomatoNet, a telediagnosis program catering to primary care dentists and physicians from southern Brazil. STUDY DESIGN: This exploratory study included all queries received by EstomatoNet from June 2015 to December 2016. Health providers (71 dentists and 18 physicians from primary care) submitted requests including clinical information and photographs of oral lesions by means of a cloud-based platform. Specialized oral medicine teleconsultants received the data, conveyed a diagnostic hypothesis, and conveyed management recommendations. RESULTS: Actinic cheilitis (n = 41, 15.8%), squamous cell carcinoma (n = 22, 8.5%), and inflammatory hyperplasia (21, 8.1%) were the most frequent diagnoses. Teleconsultants recommended referral to specialists in 42.9% of the cases, total biopsy in 23.6%, and follow-up in 16.2%. After the EstomatoNet use, the intention to refer the patients to face-to-face consultation reduced from 96.9% to 35.1%. CONCLUSION: Telediagnosis for oral lesions is feasible and has potential to improve the quality of primary health care by bridging the gap between primary and specialized health care.

In vitro bioactivity of bioresorbable porous polymeric scaffolds incorporating hydroxyapatite microspheres.

Biomimetic composites consisting of polymer and mineral components, resembling bone in structure and composition, were produced using a rapid prototyping technique for bone tissue engineering applications. Solid freeform fabrication, known as rapid prototyping (RP) technology, allows scaffolds to be designed with pre-defined and controlled external and internal architecture. Using the indirect RP technique, a three-component scaffold with a woodpile structure, consisting of poly-L-lactic acid (PLLA), chitosan and hydroxyapatite (HA) microspheres, was produced that had a macroporosity of more than 50% together with micropores induced by lyophilization. X-ray diffraction analysis indicated that the preparation and construction of the composite scaffold did not affect the phase composition of the HA. The compressive strength and elastic modulus (E) for the PLLA composites are 0.42 and 1.46 MPa, respectively, which are much higher than those of chitosan/HA composites and resemble the properties of cellular structure. These scaffolds showed excellent biocompatibility and ability for three-dimensional tissue growth of MC3T3-E1 pre-osteoblastic cells. The pre-osteoblastic cells cultured on these scaffolds formed a network on the HA microspheres and proliferated not only in the macropore channels but also in the micropores, as seen from the histological analysis and electron microscopy. The proliferating cells formed an extracellular matrix network and also differentiated into mature osteoblasts, as indicated by alkaline phosphatase enzyme activity. The properties of these scaffolds indicate that they can be used for non-load-bearing applications.

Enhanced human beta-defensin-2 (hBD-2) expression by corticosteroids is independent of NF-kappaB in colonic epithelial cells (CaCo2).

Beta-defensins are small cationic peptides with antimicrobial properties that contribute to innate host defense. Unlike human beta-defensin-1 (hBD-1), which is produced constitutively, human beta-defensin-2 (hBD-2) is expressed after adequate stimulation by cytokines and/or bacterial endotoxins in epithelial tissue and mononuclear phagocytes but may be deficient in patients with Crohn's disease. To further elucidate the role of the intestinal epithelium in antimicrobial host defense, gene regulation of hBD-2 and the interaction with NF-kappaB were analyzed in a cell culture model. Human colonic epithelial cells (CaCo2) were stimulated by pro-inflammatory cytokines (IL-1beta, TNF-alpha, IF-gamma) to induce hBD-2 mRNA transcription. Interactions with NF-kappaB were analyzed using specific inhibitors (sulfasalazine, gliotoxine, dexamethasone) at different concentrations. Defensin mRNA expression was quantified by competitive RT-PCR and antibacterial capacity of supernatants was determined by an antimicrobial assay. HBD-2 mRNA transcription and antimicrobial activity of CaCo2 cells were induced by stimulation with pro-inflammatory cytokines. Induction was not inhibited by sulfasalazine or gliotoxine, whereas dexamethasone further enhanced both gene transcription and antimicrobial capacity. The lack of inhibition of induced hBD-2 expression by specific NF-kappaB antagonists suggests an additional pathway of activation, independent of NF-kappaB. The induction of hBD-2 expression in cytokine-stimulated CaCo2 cells by corticosteroids indicates further immunomodulatory ability of steroid hormones not yet understood.

[The Indicators of Rehab Status questionnaire version 3 (IRES-3) in 1818 rehabilitees with musculoskeletal Diseases]. / Der IRES-Fragebogen Version 3 (IRES-3) bei 1818 Rehabilitanden mit muskuloskelettalen Erkrankungen.

OBJECTIVES: The revised version 3 of the questionnaire "Indicators of Reha Status" (IRES-3) was recently tested and introduced to the scientific community. The IRES-3 differs considerably from the previous version. It now comprises 16 multi-item scales that are combined to 6 dimensions and 3 single items that are combined to a 7th dimension. The aim of the study was to receive a further impression of the psychometric properties of the IRES-3 in a clinical population. METHODS: 1818 patients with musculoskeletal diseases from 10 rehabilitation hospitals completed the questionnaire at admission and at discharge. In addition to descriptive statistics (scale properties, internal consistency, sensitivity to change) logistic regression models were used to analyse the characteristics of patients in comparison to a random sample drawn from the general population. RESULTS: From a psychometric point of view the scale properties are sound. The internal consistency of the multi-item scales ranges from Cronbach's alpha = 0.77 to 0.93. The sensitivity to change ranges from SRM = 0.03 to 1.00. All scales of the IRES-3 are able to discriminate between the clinical and the general population. In all scales the clinical population demonstrated a higher level of burden than the general population. CONCLUSIONS: In our clinical population we were able to reproduce the measurement properties as reported by the authors of the IRES-3. The scales of the IRES-3 demonstrate good internal consistency and respond to change. A high discriminative validity of the instrument could be demonstrated. To further test the instrument the formation of dimensions by the authors of the IRES-3 has to be awaited.

Time course of increasing numbers of mutations in the granulocyte colony-stimulating factor receptor gene in a patient with congenital neutropenia who developed leukemia.

Point mutations in the granulocyte colony-stimulating factor receptor (G-CSFR) gene have been linked to the development of secondary leukemia in patients with congenital neutropenia (CN). This report presents data on a now 18-year-old patient with CN who has received G-CSF treatment since 1989 and who developed acute myeloid leukemia (AML) in 1998. To evaluate whether there is an association between the occurrence of point mutations of the G-CSFR gene and development of secondary AML, DNA/messenger RNA of neutrophils and mononuclear cells from this patient were analyzed at different time points by polymerase chain reaction and subsequent cloning by DNA sequencing of representative numbers of individual clones. Findings suggest an increasing instability of the G-CSFR gene in time as judged by increasing numbers of mutations proposed to be one important step in the development of AML in this patient.

Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.

Recently, point mutations in the gene of the granulocyte colony-stimulating factor (G-CSF) receptor have been reported in two patients with severe congenital neutropenia who developed acute myeloid leukemia (AML). We investigated the frequency of these specific G-CSF receptor mutations in patients with congenital neutropenia undergoing treatment with r-metHuG-CSF (Filgrastim) and the clinical relevance of these mutations. Nucleotides 2306 to 2561 including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene were amplified by reverse transcriptase-polymerase chain reaction. Detection of point mutations was performed with specific restriction enzyme analysis, as well as sequencing of PCR products. Both genomic DNA and cDNA from neutrophils and mononuclear cells were analyzed from 28 patients with severe congenital neutropenia. Four of 28 patients with congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. The point mutations replace a glutamine codon by a stop codon of the G-CSF receptor gene. Among these four congenital neutropenia patients with a mutated G-CSF receptor, two developed AML. All four patients were investigated regularly and no correlation between occurrence of G-CSF receptor mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 24 congenital neutropenia patients. Furthermore, we tested six family members of the two patients with AML including mothers and fathers, one sister, and one brother who suffers from congenital neutropenia, as well. All family members displayed a normal G-CSF receptor gene. After the acquisition of the G-CSF receptor mutations, the congenital neutropenia patients continued to respond to G-CSF therapy with an increase in absolute neutrophils in the peripheral blood. We conclude that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur spontaneously and are not inherited. From our data, we suggest that the described G-CSF receptor point mutations do not alter the response to treatment with r-metHuG-CSF and are not the cause of severe congenital neutropenia.

Frequency of point mutations in the gene for the G-CSF receptor in patients with chronic neutropenia undergoing G-CSF therapy.

Point mutations in the gene for the G-CSF receptor have been reported previously in a subgroup of patients with severe congenital neutropenia. Here, we investigated the frequency of these specific G-CSF receptor mutations in patients with neutropenic disorders undergoing treatment with recombinant human (r-metHu)G-CSF (Filgrastim). Nucleotides 2306 to 2561, including the critical region (nucleotides 2384-2429) from the intracellular domain of the G-CSF receptor gene, were amplified by reverse transcriptase-polymerase chain reaction, and DNA was sequenced directly and after transformation in E. coli. Four of 30 patients with severe congenital neutropenia displayed a point mutation in the tested cytoplasmic region of the G-CSF receptor gene. Two of the four patients with a mutated G-CSF receptor developed acute myeloid leukemia secondary to congenital neutropenia. G-CSF receptor analyses were performed in myeloid cells taken at different time points in the four patients with the mutated receptor, and no correlation between occurrence of the mutation and time or dose of r-metHuG-CSF treatment was found. No point mutations in the G-CSF receptor critical domain could be detected in cells from the other 26 congenital neutropenia patients. Additionally, no G-CSF receptor point mutations could be seen in neutrophils, blood and bone marrow mononuclear cells from patients with cyclic or idiopathic neutropenia, and bone marrow mononuclear cells from patients suffering from severe aplastic anemia. Similar results were obtained by Touw et al., demonstrating that five out of 25 patients with congenital neutropenia reveal G-CSF receptor mutations. These data show that the point mutations in the critical region of the intracellular part of the G-CSF receptor occur only in a subgroup of severe congenital neutropenia patients. Furthermore, our data suggest that the described G-CSF receptor point mutations are not correlated with the start, duration or doses of r-metHuG-CSF treatment, but might result from genetic instability in the G-CSF receptor gene in severe congenital neutropenia.

[Adrenomyeloneuropathy. A frequent cause of Addison's disease]. / Adrenomyeloneuropathie. Eine häufige Ursache des Morbus Addison.

Adrenomyeloneuropathy (AMN) is a "milder form" of adrenoleukodystrophy with a X-linked inheritance. Abnormal catabolism of the very long-chain fatty acids (VLCFA) results in Addison's disease and spastic paraparesis. The VLCFA concentration was measured in 23 of 26 patients with Addison's disease (mean age 48.5 [20-75] years) being treated at the University Hospital Marburg during May, 1991. The concentration was elevated in four of the 12 men with the disease, while it was within normal limits in the 11 women. Only two patients had paraparesis-like neurological deficits. This finding suggests that AMN is not as rare as has been supposed. It is recommended that the concentration of VLCFA be measured in all patients with Addison's disease, because an increase could have important consequences.

Dehydroepiandrosterone (DHEA) levels in patients with prostatic cancer, heart diseases and under surgery stress.

DHEA levels in patients with prostatic cancer were significantly lower, but total and free testosterone (T) significantly higher as those in an age-matched control group. Therefore, the calculated quotients DHEA/free T and DHEA/T were especially different between both groups. DHEA and DHEAS levels in patients with heart diseases were also significantly lower but cortisol (F) levels were significantly higher as those in a control group. The quotients DHEA/F and DHEAS/F were also of greater significance between both groups than the hormone values alone. The response of DHEA and F levels in patients undergoing surgery showed an increase of both steroids under surgery. On the second postoperative day, however, F levels were still significantly higher but DHEA levels were significantly lower as the initial values. The differences between the initial values and those on the second postoperative day of F and DHEA showed a significant correlation, i.e. the higher the elevation of F levels above the initial values the greater was the diminution of DHEA levels below the initial values.

[Determination of Legionella antigens using monoclonal antibodies]. / Bestimmung von Legionella-Antigenen mit Hilfe monoklonaler Antikörper.

The detection of antigens is the most important tool for rapid diagnosis of Legionellosis. 34 Legionella (L.) spp. with 51 serogroups have been identified from several sources. According to the antigenic diversity, it is necessary to select monoclonal antibodies (mab) adequate to diagnostic purposes. Mab with specificity to genus, species and serogroups were discussed. L. pneumophila accounts for 70 to 80% of all cases of Legionelloses. In this study self-made mab to L. pneumophila are presented that demonstrate these bacteria in clinical materials from respiratory tract using immunofluorescent tests and by detection of soluble antigens in pleura fluids and urine specimens using enzyme immunoassay.

Monoclonal antibodies to Legionella pneumophila serogroup 6: evidence of antigenic diversity.

Reactivity of monoclonal antibodies (Mabs) prepared against the type strain of Legionella pneumophila subsp. pneumophila serogroup 6 (Lp sg 6) was tested in the indirect immunofluorescence test using 16 environmental isolates of this sg and 58 strains of sg 1 to 5 and 7 to 14. Five out of 11 Mabs 5 were serogroup-specific, i.e. there was a reaction with all sg 6 strains tested, but not with strains from other sg. Further 4 Mabs reacted with all sg 6 strains and a few strains of other sg. Two Mabs were only reactive with the type strain of Lp sg 6 and one sg 6 strain isolated in Bratislava, Czechoslovakia. This report shows further evidence that Lp sg 6 can be divided into antigenically distinct subtypes.

Common epitope on urinary antigen derived from different Legionella pneumophila serogroup 1 strains recognized by a monoclonal antibody.

Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

[Detection of legionellas in clinical samples using FITC-marked antibodies]. / Nachweis von Legionellen in klinischen Untersuchungsmaterialien mit FITC-markierten Antikörpern.

Using serogroup/species specific FITC-conjugates against Legionella (L.) pneumophila serogroups (SG) 1 to 6, L. micdadei, L. bozemanii and L. jordanis 186 sputum samples, 43 pleural fluids and bronchial washings and 39 lung tissue samples from 251 pneumonia patients were checked for Legionellae with the direct fluorescent antibody technique (DFAT). In samples from 201 patients which were negative if tested for antibodies and for urinary antigen and/or using culture methods, the DFAT was negative, too. One Pseudomonas strain isolated from a bronchial washing showed crossreactions. In 19 out of 49 patients with legionellosis L. pneumophila SG 1 (3x), SG 2 (1x), SG 3 (4x), SG 4 (1x), SG 5 (5x), L. micdadei (3x) and L. bozemanii (2x) were found. Positivity of the DFAT was significant higher in specimens, taken in early stages of illness (p less than 0.01). DFAT can be used for specific detection of Legionellae in clinical specimens, but the relative low sensitivity (39%) does not allow to exclude legionellosis. Further diagnostic tests like detection of antibodies and urinary antigen and culture are necessary to diagnose legionellosis.

Radioimmunological characterization of carcinoembryonic antigen (CEA) preparations.

A multi-step radioimmunological test system was applied to the characterization of carcinoembryonic antigen (CEA) preparations obtained by column chromatography including ion exchange procedure. CEA contents were estimated by means of a sequential inhibition radioimmunoassay using Protein A-bearing Staphylococci for coprecipitation. Data were expressed as "units CEA" and compared with results obtained from commercial CEA-RIA kits. In addition, attempts were made to evaluate the presence of normal cross-reacting antigens in CEA preparations by the application of an antiserum to perchloric acid extract from normal lung tissue. Data from these tests were expressed as "units NLA" (NLA stands for "normal cross-reacting lung antigen"). The factor "units CEA/units NLA" proved useful as an indication of the tumour specificity of those antigenic components measured in the inhibition assay. Moreover, radioimmunoprecipitation tests with subsequent SDS polyacrylamide gel electrophoretic analysis were performed in order to get information on the molecular weight of the molecule(s) involved in the radioimmunological CEA determination procedure. The test system displayed 1) highly specific CEA reagibility in the standard inhibition assay, 2) test sensitivity of about 200 pg CEA (when related to the Hoffman/La Roche test) and 1 ng CEA (when related to CEA-IRE-SORIN), 3) low reagibility to normal cross-reacting antigens, also when compared to the commercial tests, 4) a molecular weight of about 200,000 daltons for the component(s) measured, and 5) evidence of inhomogeneity of the CEA batches investigated and those international references available. The latter might in part be attributed to so-called species-specific CEA-related determinants. The test system proved also useful for CEA estimations in crude tissue extracts.

Is there any diagnostic relevance of human antibodies which react with mouse mammary tumor virus (MuMTV)?

Applying an indirect immunofluorescence assay with prolonged serum incubation on frozen-cut mouse mammary tumor slices, antibodies are detectable in human sera which react with viral inclusion bodies consisting of intracytoplasmic A particles (iAp). These iAp are known to represent intracellular entities of the mouse mammary tumor virus (MuMTV), mainly related to MuMTV core constituents. In 1,449 women studied, the antibody incidence is partially correlated with proliferating changes of the mammary gland. However, epidemiological data obtained thus far have clearly shown that the detection of antibody is not useful in breast cancer diagnosis (antibody incidence in breast cancer patients 22.8%, in lactating women 20.6%, in controls 5.9%). Studying antibody reagibility to particular iAp polypeptides by means of a radioimmunoprecipitation (RIP) technique, there has been preliminary evidence that human serum factors react with more than one mouse virus protein. The most prominent reactivity was seen to be directed to the 14,000 dalton protein (Ap14). The detection of serum antibody activity is considered as being of important indicative value in that it reflects the existence of MuMTV-related antigens in man.