Extrinsic allergic alveolitis: comparative study of the bronchoalveolar lavage profiles and radiological presentation.

INTRODUCTION: Extrinsic allergic alveolitis (EAA) is an immunologically mediated interstitial lung disease. The abnormalities in the bronchoalveolar lavage (BAL) fluid cell counts are almost always seen in patients with EAA according to the stage of the disease. The aim of this retrospective study was to find out how the BAL lymphocyte count, percentage of lymphocytes expressing HLA-DR, CD4/CD8 T cell ratio in BAL fluid, and the concentration of immunoglobulin G in serum correspond to the inflammatory activity of the disease. METHODS: The study included 14 patients with EAA. BAL fluid samples were obtained and processed for cytological and cytometric analysis. Immunoglobulin G serum concentrations were measured. High resolution computed tomography (HRCT) scoring system modified by Gay was used for establishing the alveolar and interstitial score in each patient. CONCLUSIONS: It was found that subjects with normal value of CD4/CD8 ratio in BAL fluid had higher interstitial HRCT score. Clinical presentation, continuous exposure to the causative antigens, and BAL lymphocyte count positively correlated with the alveolar HRCT score. It is proposed that the increased BAL lymphocyte count could be the predictor of the inflammatory activity of the disease, especially in people with lasting exposure to the offending antigen.

[Viability of parathyroid gland tissue measured by flow cytometry]. / Viabilita tkáne prístítných telísek merená prutokovou cytometrií.

BACKGROUND: Postoperative hypoparathyroidism after the total parathyroidectomy (PTX) remains a problem, no matter our experiences with 243 operations on parathyroid glands (PG). Implantation of "fresh" tissue, cryopreservation and reimplantation of cryopreserved tissue are performed with uncertain results. The aim of this project was to compare viability of cryopreserved tissue of parathyroid glands with "fresh" tissue obtained during parathyreoidectomy and with tissue from cadaverous donors. METHODS AND RESULTS: Group 1 included 55 cryopreserved samples obtained from 41 patients after PTX (22M, 19F, a mean age of 46 +/- 11 years). Average duration of storage in liquid nitrogen was 84 +/- 49 months. Group 2 included "fresh" tissue of PG, harvested during PTX. Viability was measured in different time in samples from 42 patients with hyperparathyroidism (11M, 31F, a mean age of 55 +/- 13 years). Group 3 included tissue of 14 cadaverous donors obtained during multiorgan harvesting (7M, 7F, a mean age of 31 +/- 5 years, WIT 32 min). Viability was measured by flow cytometry with propidium iodide after dissociation of tissue. Evaluation of PG tissue was proven by histology. Average viability in group 1 was 36.9 +/- 24.7%, no correlation with the duration of storage in liquid nitrogen was found. Average viability in group 2 was 51.4 +/- 24%. Viability in group 3 was 66.8 +/- 32%. Group 1 vs. group 2 were different with p < 0.05, group 2 vs. 3 did not reach significance (with marginal p = 0.06) and group 1 vs 3 were different with p < 0.001. CONCLUSIONS: The highest viability was found in tissue of cadaverous donors, the lowest in cryopreserved tissue (with no correlation to the duration of storage in liquid nitrogen).

Cd38 expression on Cd8+ T cells in Human immunodeficiency virus 1-positive adults treated with HAART.

The aim of this study was to assess whether the density of CD38 antigen expression on CD8+ T cells can be used as a marker of activation of the immune system in Human immunodeficiency virus 1 (HIV-1)-positive patients treated with highly active antiretroviral therapy (HAART). T cell subsets, expression of CD38 antigen on CD8+T cells, HIV-1 viral load and stage of the disease were analyzed at baseline and after 12 months of HAART in 24 HIV-1-infected patients. Our data showed that the use of HAART is effective in reducing plasma viral load and in achieving a stable CD4+ count and percentage of CD8+/CD38+ cells. The percentages of CD8/CD38+ cells in HIV-1-infected patients at baseline and after 12 months of HAART were significantly higher than those of controls. Analysis of the density of CD38 expression revealed that it was due to CD8+/CD38+ subsets with low and medium density of antigen expression. Absolute number of CD4+ T cells correlated negatively with the percentage of CD8+/CD38+ cells at baseline of the study. Persistent up-regulation of the CD38 expression on CD8+ T cells and its correlation with the decreased CD4+ count despite the reduction of plasma viral load may reflect residual replication of HIV-1 in reservoirs. Thus, this immunological parameter can serve as a biological marker of HIV-1 infection and might have utility in clinical management of HIV-1-infected persons.

[Analysis of the inflammation reaction and selected indicators of immunity in patients with an infected diabetic ulcer]. / Analýza zánetlivé odpovedi a vybraných ukazatelu imunity u pacientu s infikovanými diabetickými ulceracemi.

BACKGROUND: Patients with diabetes represent 50 to 70% of patients who undergo nontraumatic foot or leg amputation, caused mostly by infection and necrosis of soft tissues accompanied with osteomyelitis. Signs and symptoms of infections may often be absent in patients with infected foot ulcers--the syndrome of "diabetic foot" (DF). The course and consequences of the infection may be influenced by the immune system dysfunction. The aim of our study was to assess presence of the chronic inflammation and specific immune responses, both humoral and cellular in patients with diabetic foot infection. METHODS AND RESULTS: 34 patients treated over one month for an infected DF in our foot clinic (mean age 54 +/- 8 years, mean duration of diabetes 20 +/- 9 years, mean HbAlc 8.8 +/- 1.5%), were matched with 27 healthy subjects. All patients were without clinical signs of acute deep foot infection and without critical leg ischemia. The inflammatory response was assessed by white blood cells count and C-reactive protein (CRP), humoral immune response was assessed by immunoglobulins (Ig) and cellular immunity was evaluated by T lymphocytes subpopulations. Patients with DF compared with healthy controls exhibited the laboratory signs of infection--significantly increased white blood cells count (7.6 +/- 2.1 vs. 6.4 +/- 1.3.10(9)/l, p < 0.01) and neutrophil count (4.6 +/- 1.8 vs. 3.8 +/- 0.9.10(9)/l, p < 0.05) and significantly increased CRP (7 +/- 12 vs. 2 +/- 6 mg/l, p < 0.01). Patients with DF had also significantly higher IgA levels (3.5 +/- 1.6 vs. 2.7 +/- 1.1 g/l, p < 0.05) and significantly more CD3+ T cells (76 +/- 8 vs. 71 +/- 10%, p < 0.05) and suppressor/cytotoxic CD8+ T cells (32 +/- 11 vs. 26 +/- 10%, p < 0.05). Other followed parameters IgG, IgM and serum monocyte and lymphocyte counts, CD4+ helper T cells and CD4+/CD8+ T-cell ratio did not differ between patients with DF and healthy controls. CONCLUSIONS: We did not anticipate a severe secondary immunodeficiency in followed cellular and humoral immune parameters in patients with chronic bacterial foot infection. It is necessary to assess the sufficiency of immune system activation with respect to chronic inflammation in next research.

[Non-specific immune responses in patients with chronic diabetic foot syndrome and chronic bacterial infection]. / Nespecifické mechanismy imunity u pacientu se syndromem diabetické nohy s chronickou bakteriální infekcí.

Chronic bacterial ulcers infection is a frequent cause of non-healing diabetic foot. The major factors of a non-specific immune response are phagocytic cells including polymporphonuclear (PMN) leukocytes, and humoral systems (complement). PMN leukocytes remove microorganisms by phagocytosis a part of it is intracellular killing and degradation in a process requiring energy and associated with "respiratory burst". The aim of our study was to assess non-specific immune response in patients with diabetic foot syndrome and chronic bacterial infection. 30 patients treated over one month with antibiotics for an infected diabetic foot in our foot clinic had significantly lower values of "oxidative burst" of PMN leukocytes in basal state (396 +/- 228 vs. 574 +/- 337, p < 0.05) in comparison with 25 matched healthy controls. There were no significant differences neither in the count of active phagocyting PMN leukocytes and their initial phagocytic activity nor in the humoral component of non-specific immunity (in circulating immunocomplexes, C3 and C4 components of complement) between both groups. The results of our study show a slightly altered non-specific immune response in patients with diabetic foot syndrome and chronic bacterial infection.

Cell-cell contacts with epithelial cells modulate the phenotype of human macrophages.

Interactions of macrophages with epithelium represent one of the pathways involved in regulating local immune mechanisms. We studied the effect of cell-cell contact with an epithelial monolayer on the phenotype of macrophages. Human monocytes and THP-1 macrophages were co-cultured with monolayers of human bronchial epithelial cells (HBECs), the alveolar type II-like cell line A549, renal adenocarcinoma epithelial cells (RA), and the lung fibroblast strain HFL-1. The expression of CD11b, CD14, CD54, and HLA-DR was measured by immunocytochemistry and flow cytometry and showed epithelial cell induction of CD54 and HLA-DR in monocytes and of all antigens in THP-1 cells. Co-culture with fibroblasts did not change the phenotype of macrophages. Separation by a filter insert inhibited most of the effects. Culture supernatants did not induce prominent phenotypic changes. Cell-cell contacts with epithelium appear to be of importance in regulating the phenotype of macrophages.

[Changes in cellular immunity after administration of nucleotides]. / Zmeny celulární imunity po podání nukleotidu.

BACKGROUND: Nucleotide deficiency in the diet of experimental animals reduces cellular immunity, and conversely it is assumed that an increased intake could improve the immunity also in humans. METHODS AND RESULTS: The authors administered to 12 women with malignant tumours the dietetic preparation Preventan containing nucleotides, 1 x 1 mg/day, and followed up for four weeks some immunological parameters. There was a considerable decline of CRP and conversely an increase in the number of T-lymphocytes (CD3+), T-lymphocyte helpers (CD4+) and suppressor/cytotoxic T-lymphocytes (CD8+), almost to double values. There was also an increase of the cytotoxic activation and activated T-lymphocytes. As to parameters of humoral immunity, except for a small rise of IgG, no major changes were recorded and the number of B-lymphocytes did not change (CD19). CONCLUSIONS: Supplementation with a dietetic preparation containing nucleotides increases the number of T-lymphocytes, their sub-populations and their activation, and thus has a favourable impact on cell-mediated immunity as apparent from the marked decline of CRP.

Flow cytometry cross-match before kidney transplantation in relation to early postransplant rejection.

The aim of this study was to compare flow cytometry cross match (FCXM) results in patients before first kidney transplantation with the incidence of rejection episodes and kidney graft survival after transplantation. Sera of 51 patients obtained immediately before transplantation were tested on spleen cells of respective kidney donors. We found no correlation between a positive FCXM result before transplantation and the occurrence of immunological complications after transplantation.

[Genetic diagnosis of phenylketonuria. IV. Mutations of phenylalanine hydroxylase gene in Caucasian and Gypsy populations in Czech and Slovakia Republics].

Direct sequencing was conducted on the regions of the exon 7 and 12 in the phenylalanine hydroxylase (PAH) gene amplified by the polymerase chain reaction, using solid-phase technology involving the biotin streptavidin system. A novel mutation and seven previously known mutations were identified in the PAH genes among 15 Caucasians and 10 Gypsies in the Czech and Slovakia republics, affected with classical phenylketonuria (PKU). Two of these substitutions (R243X and G272X) resulted in the generation of a premature stop codon, and a single base transition of G to A at codon 261 resulted in the substitution of Arg for Gln (R261Q). These three mutations together accounted for 16.7% of PKU alleles among 15 Caucasians. The R252W mutation was detected in these two groups: two Caucasians were compound heterozygous for the P281L or R408W mutations (6.7% of all mutant alleles). However, all 10 Gypsy PKU patients were homozygous for the R252W transition. The R408W mutation accounted for 43% of PKU alleles in 15 Caucasian PKU patients. A novel heterozygous C-to-G transversion at the third base of codon 263 in the exon 7, resulting in the substitution of Phe for Leu (F263L), was detected in a Caucasian PKU patient (3.3% of all mutant alleles). In this study, we revealed a novel PKU mutation of the F263L in Caucasian populations and a high-risk PKU mutation of the R252W in Gypsy populations.

Gypsy phenylketonuria: a point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia.

A direct mutational analysis of the phenylalanine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. We analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 "classical PKU" patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group we detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uninformative in the determination of heterozygosity. ASO hybridization served us effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects.

[Genetic diagnosis of phenylketonuria: identification of the mutations of phenylalanine hydroxylase gene by PCR direct sequencing].

To investigate the mutations of the phenylalanine hydroxylase (PAH) gene in Orientals, direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid phase technology involving the biotin-streptavidin system. Four mutations possibly associated with phenylketonuria (PKU) were identified in a Chinese and four Japanese patients. A novel Arg158 (CGG)-to-Trp158 (TGG) mutation was identified in exon 5 of the PAH gene in a Chinese PKU patient. The second change was due to a G-to-A transversion at the last base of intron 4. The third change was a compound heterozygote; one mutation was a G-to-A transversion at the last base of intron 4. The other was a G-to-C transversion at the second base of codon 413, which resulted in a substitution of Arg(CGC) by Pro(CCC) in exon 12. The last change was due to a Tyr204(TAT) -to-cys204(TGT) mutation in exon 6 of the PAH gene in two Japanese. This preliminary study revealed a novel PKU mutation and considerable genetic heterogeneity in the PAH gene among Orientals.