The Clinical and Molecular Assessment of Iranian Families with Severe Congenital Neutropenia, Identification of HYOU1 and SHOC2 as Potential Novel Gene Defects.

Neutropenia congenita grave (SCN) is a rare disease with a genetically and clinically heterogeneous nature, usually diagnosed in childhood, with an elevated risk of infections such as otitis, skin infections, pneumonia, deep abscesses, and septicemia. Patients with SCN also have an increased risk of leukemia, and mutations in the ELANE and the HAX1 genes have been observed in those patients. This study was conducted to genetically screen six Iranian families with SCN who have at least one affected person. In the first step, all exons and intron boundaries of ELANE and HAX1 genes were sequenced in probands. Cases with no pathogenic mutations were tested through whole-exome sequencing (WES). Analysis showed five different variants in ELANE (c.377 C>T), HAX1 (c.130_131 insA), HYOU1 (c.69 G>C and c.2744 G>A) and SHOC2 (c.4 A>G) genes in four families. We found that two out of six families had mutations in ELANE and HAX1 genes. Moreover, we found two novel mutations at the HYOU1 gene that had not previously been reported, as well as a pathogenic mutation at SHOC2 with multiple phenotypes, that will contribute to determining the genetic basis for SCN. Our study revealed that WES could help diagnose SCN, improve the classification of neutropenia, and rule out other immunodeficiencies such as autoimmune neutropenia, primary immunodeficiency diseases, and inherited bone marrow failure syndromes.

Comparison of 18F-FDG PET-MR and fecal biomarkers in the assessment of disease activity in patients with ulcerative colitis.

OBJECTIVE: To compare the diagnostic performance of fecal biomarkers and 18F-fludeoxyglucose (18F-FDG) positron emmision tomography-MR (PET-MR) in the assessment of disease activity in patients with ulcerative colitis. METHODS: This study was conducted under the framework of a single-center clinical trial (clinicaltrials.gov [NCT03781284]). N = 50 participants were enrolled. Fecal samples were collected before bowel preparation. All patients underwent whole-body 18F-FDG PET-MR followed by ileocolonoscopy within 24 h. Diagnostic performance of five fecal biomarkers (calprotectin, lactoferrin, polymorphonuclear leukocyte elastase, S100A12 and eosinophil-derived neurotoxin), MR morphological parameters (MRmorph), diffusion-weighted imaging and PET in detecting active disease determined by Rachmilewitz endoscopic activity index (EAI) were evaluated and compared with each other. Correlations between fecal biomarkers, PET and endoscopy were calculated. RESULTS: According to EAI, n = 38 patients presented with endoscopically active disease (16 mild, 19 moderate and 3 severe). All five biomarkers, PET and MRmorph could differentiate endoscopically active disease from endoscopic remission without significant difference regarding their operating characteristics (accuracies between 0.673 for calprotectin and 0.898 for lactoferrin). In predicting endoscopically moderate to severe disease, PET showed the highest diagnostic performance (accuracy = 0.857) compared to calprotectin and lactoferrin (accuracy = 0.633 and 0.735). PET had also the strongest correlation with endoscopy (ρ = 0.685, p < 0.001), while within fecal biomarkers the levels of lactoferrin and eosinophil-derived neurotoxin correlated significantly with EAI (ρ = 0.423 and 0.528, both p < 0.05). CONCLUSION: Both fecal biomarkers and PET-MR were excellent non-invasive diagnostic tools in the assessment of disease activity in ulcerative colitis. ADVANCES IN KNOWLEDGE: Both fecal biomarkers and PET-MR parameters are able to predict endoscopically active disease with comparable diagnostic performance. PET had the highest correlation with endoscopy and outperformed fecal biomarkers in differentiating moderate to severe from mild disease.

Magnetic resonance imaging assessment of proteolytic enzyme concentrations and biologic properties of intraluminal thrombus in abdominal aortic aneurysms.

OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and ß-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Assessment of systemic inflammation by time-trends of blood granulocyte count and plasma myeloperoxidase and elastase concentrations following colic surgery in horses.

OBJECTIVE: To determine changes in blood granulocyte counts and in plasma myeloperoxidase (MPO) and elastase (ELT) concentrations in surgical colic cases, and to determine the relationship between these changes and the surgical procedure performed, occurrence of postoperative ileus, and final outcome. DESIGN: Prospective clinical study conducted over a 12-month period. SETTING: University teaching hospital. ANIMALS: Fifty-three horses undergoing emergency laparotomy and surviving at least 12 hours postoperatively. INTERVENTIONS: Blood samples were taken before surgery, during surgery, at the recovery from anesthesia, and then serially until the 150th hour after the first blood sampling. Granulocyte counts were performed by an automated cell hematology analyzer. Specific ELISAs were performed for the MPO and ELT measurements. Mixed models were used to compare the time-trends of the 3 parameters. MEASUREMENTS AND MAIN RESULTS: Taking all horses together, the time-trends of MPO and ELT were not significantly different from each other, but they were significantly different from the granulocyte time-trend. The type of surgical procedure did not influence the time-trends of the 3 parameters. Significant changes in the granulocyte time-trends were associated with postoperative ileus and outcome. Significant changes in the MPO time-trends were associated with outcome. The ELT time-trends were not influenced by ileus or outcome. CONCLUSIONS: Granulocyte counts and MPO change over time and are related to the severity of the inflammatory reaction in surgical colic cases. These time-trends may allow evaluation of treatment efficacy in an effort to modulate excessive granulocyte activation and degranulation.

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.

BACKGROUND: During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin. METHODS: Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40). RESULTS: Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls. CONCLUSIONS: The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

[Possibilities of the use of immunological indicators for the assessment of the risk of manifestation of endogenous psychoses in patients with nonpsychotic disorders of the juvenile age].

OBJECTIVE: To study parameters of innate and adaptive immunity in the blood serum of patients with nonpsychotic mental disorders and to classify them by risk of psychosis manifestation. MATERIAL AND METHODS: Authors studied 49 male patients, aged from 16 to 25 years, with nonpsychotic mental disorders corresponded to the premanifest stage of endogenous psychosis. The activity of leukocyte elastase (LE), functional activity of alpha-1-proteinase inhibitor (α1-PI) and the level of autoantibodies (aAB) to S-100 and basic myelin protein were measured. RESULTS: A significant increase in LE and α1-PI was found in patients compared to controls (p<0.001). The level of aAB to neuroantigens was similar in patients and controls. The increase in LE activity was positively correlated with HAM-D depressive symptoms and SOPS total scores (r=0.47, p=0.02). Correlations between α1-PI activity and scores on SOPS positive subscale (r= -0.61, p=0.002) and SOPS total scores (r= -0.43, p=0.04) were identified. After treatment, the improvement of patient's state assessed by SOPS and HAM-D was correlated with the decrease in LE activity in 80% (p<0.01). The further increase of LE activity in 20% may be considered as an indicator of low quality remission and risk of psychosis manifestation. CONCLUSION: Patients with nonpsychotic mental disorders with higher levels of inflammation markers may be attributed to high risk group.

Imbalance between neutrophil elastase and its inhibitor α1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.

Peritonitis from perforated appendicitis: stress response after laparoscopic or open treatment.

Elevated intra-abdominal pressure during laparoscopy may promote systemic inflammatory response. In patients with generalized peritonitis from perforated appendicitis, we sought to compare acute phase response and immunologic status from laparoscopic and open approach. One hundred and forty-seven consecutive patients underwent appendectomy for perforated appendicitis (73 patients had laparoscopic appendectomy and 74 patients had open appendectomy. Bacteremia, endotoxemia, white blood cells, peripheral lymphocytes subpopulation, human leukocyte antigen-DR (HLA-DR), neutrophil-elastase, interleukin-1 and 6 (IL-1 and 6), and C-reactive protein were investigated. One hour after intervention, bacteremia was significantly higher in the open group compared with the laparoscopic group (P < 0.05). A significantly higher concentration of systemic endotoxin was detected intraoperatively in the open group of patients in comparison with the laparoscopic group (P < 0.05). Laparotomy caused a significant increase in neutrophil concentration, neutrophil-elastase, IL-1 and 6, and C-reactive protein and a decrease of HLA-DR. We recorded 6 cases (8.1%) of intra-abdominal abscess in the open group and one (1.3%) in the laparoscopic group (P < 0.05). Open appendectomy, in case of peritonitis, increased the incidence of bacteremia, endotoxemia, and systemic inflammation compared with laparoscopic appendectomy. Early enhanced postoperative systemic inflammation may cause lower transient immunologic defense after laparotomy (decrease of HLA-DR), leading to enhanced sepsis in these patients.

A review of augmentation therapy for alpha-1 antitrypsin deficiency.

INTRODUCTION: Alpha-1 antitrypsin deficiency (AATD) is a relatively common, but under-recognized condition which manifests commonly with liver cirrhosis and emphysema. Specific therapy for lung-affected individuals with AATD is augmentation therapy, which consists of intravenous infusion of purified human plasma-derived alpha-1 antitrypsin (AAT). Augmentation therapy was first approved by the United States Food and Drug Administration (FDA) in 1987 for emphysema associated with severe AATD and today, six augmentation therapy preparations, all of which derive from pooled human plasma, have received FDA approval. AREAS COVERED: This paper reviews augmentation therapy for AATD, including the various available commercial preparations, their processing and biochemical differences, evidence regarding biochemical and clinical efficacy, patterns of clinical use, adverse effect profiles, cost-effectiveness and potential uses in conditions other than emphysema associated with AATD. Novel and emerging strategies for treating AATD are briefly discussed next, including alternative dosing and administration strategies, recombinant preparations, small molecule inhibitors of neutrophil elastase and of AAT polymerization, autophagy-enhancing drugs and gene therapy approaches. EXPERT OPINION: We conclude with a discussion of our approach to managing patients with AATD and use of augmentation therapy.

Predictive relevance of clinical scores and inflammatory parameters in secondary peritonitis.

HYPOTHESIS: To measure and evaluate clinical scores and various inflammation parameters for providing a better outcome assessment of patients with secondary peritonitis. DESIGN: Prospective study. SETTING: ICU of a university and a university affiliated hospital. PATIENTS: Fifty-six patients with severe secondary peritonitis were enrolled in this study executed within 4 years. MEASUREMENTS AND MAIN RESULTS: Blood samples were taken preoperatively and 2, 6, 8, 12, 18, 24, 30, 36, 42 and 48 hours post operation, thereafter every 12th hour until day 5 respectively once daily until day 14. Etiology of peritonitis, clinical score systems (APACHE II, MOF and SOFA), and 27 mainly with activity tests or enzyme-immunoassays measurable inflammation parameters were simultaneously analyzed and stratified into lethal outcome (n = 11) or survival (n = 45), respectively. The etiological distribution of peritonitis was identical among both groups. Proportion of intraperitoneal fungi, E. coli, and bacteroids was substantially higher during the primary operation in the group with lethal outcome. With increasing significance initial and follow-up APACHE II, MOF and SOFA scores provided higher values in this group. Various plasma/serum parameters of hemostasis, leukocyte proteolytic system, acute phase reaction, cytokine system, cell adhesion, opsonization, and main organ functions showed significantly different values between both groups at the preoperative stage and/or during observation period I (day 0-4). Logistic regression analysis revealed the SOFA score and neopterin concentration as the combination with the best sensitivity (63.6%) and specificity (93.2%) for predicting the patients' survival even at the preoperative stage. For the observation period I, the combination of SOFA score and TNF receptor II showed the highest predictive sensitivity (72.7%) and specificity (95.6%). CONCLUSION: Evaluation of the severity of secondary peritonitis using a scoring system with high prognostic relevance could conceivably result in an earlier and adequate application of intensive care such as hemofiltration, administration of immunoglobulins and serial abdominal lavage to improve successful outcome.

[The assessment of neutrophil elastase measurements usefulness in pregnant women with premature rupture of fetal membranes and chorioamnionitis suspicion]. / Ocena przydatnosci oznaczania elastazy neutrofilowej u ciezarnych z przedwczesnym peknieciem blon plodowych w przypadkach podejrzanych o subkliniczna infekcje wewnatrzowodniowa.

OBJECTIVES: The evaluation of neutrophil elastase (NE) levels and its usefulness in pregnant women with premature rupture of foetal membranes (PROM) and chorioamnionitis suspicion. MATERIAL AND METHODS: We evaluated the relationship between maternal plasma and amniotic fluid NE levels with the presence of chorioamnion infection in sixty pregnant women, divided into two groups--with and without PROM. The diagnostic performance of NE evaluations in discrimination of suspected intraamniotic infection was calculated. RESULTS: NE levels in PROM patients are significantly higher than in the control group (p < 0.000001). Significantly higher NE concentrations are also observed in the case of chorioamnionitis. Moreover, if at least two clinical markers of infection were present, the diagnostic value of amniotic fluid NE levels proved to be 100% sensitive and of 100% negative predictive value. CONCLUSIONS: NE levels may be used as clinical markers which enable the obstetricians to exclude chorioamnionitis.

Coated prostheses are associated with prolonged inflammation in aortic surgery: a cost analysis.

This prospective study was conducted to compare inflammatory responses between patients receiving coated and uncoated vascular prostheses, and to examine their effect on length of stay and cost of patients undergoing abdominal aortic aneurysmectomy. Patients undergoing elective vascular reconstruction of an abdominal aortic aneurysm were assigned randomly to coated-graft or uncoated-graft groups (n = 20, for each group). Interleukin (IL)-6, granulocyte elastase, white blood cell count, C-reactive protein (CRP), and body temperature (BT) were prospectively recorded preoperatively and on postoperative days (PODs) 1, 3, 7, and 14. In-hospital stay and hospitalized costs were also analyzed. IL-6 and CRP concentrations in the coated-graft group were higher than those in the uncoated-graft group (P = 0.01 and 0.05). BT was more frequently elevated >37 degrees C at POD 14 in the coated-graft group than in the uncoated-graft group (P =0.03). Discharge was delayed, and overall hospitalization cost was higher in the coated-graft group than in the uncoated group (17.6 vs. 13.5 days, and 2 010 000 vs. 1 780 000 yen, P = 0.006 and P = 0.002, respectively). Coated vascular prosthesis demonstrated more profound inflammatory reaction than noncoated prosthesis, postoperatively.

Noninvasive markers in the assessment of intestinal inflammation in inflammatory bowel diseases: performance of fecal lactoferrin, calprotectin, and PMN-elastase, CRP, and clinical indices.

OBJECTIVES: The aim of this study was to compare the performance of fecal lactoferrin (Lf), calprotectin (Cal), polymorphonuclear neutrophil elastase (PMN-e), as well as serum C-reactive protein (CRP) in patients with inflammatory bowel diseases (IBD) to address (a) whether these markers can differentiate IBD patients with endoscopically assessed inflammation from IBD patients without inflammation and from irritable bowel syndrome (IBS); (b) whether they correlate with endoscopic severity of inflammation; and (c) whether a combination of fecal markers with the respective disease-specific activity indices may increase the diagnostic accuracy with reference to the endoscopic severity of inflammation. METHODS: Fecal levels of Lf, Cal, and PMN-e and serum CRP were assessed in 139 patients undergoing diagnostic ileocolonoscopy (54 IBS patients, 42 ulcerative colitis [UC], 43 Crohn's disease [CD]). Disease activity was determined for CU with the colitis activity index (CAI) and for CD with the Crohn's disease activity index (CDAI). The performance of each marker with reference to endoscopic inflammatory activity was assessed by computing correlations, and sensitivity and specificity using published as well as adjusted cutoffs. A comprehensive activity index was computed by combining results from fecal markers, serum CRP, and a clinical activity index. RESULTS: UC or CD patients with active inflammation demonstrated significantly higher levels of Lf, Cal, and PMN-e in feces as well as serum-CRP when compared to patients with inactive inflammation as well as patients with IBS (all P < 0.05). Using adjusted cutoffs enabled a marked improvement of all markers with an overall diagnostic accuracy in IBD of 80.0% for Lf, 80.0% for Cal, 74.1% for PMN-e, 64.0% for CRP, and 79.0% for the respective clinical disease scores. Cal showed the highest diagnostic accuracy in CD (81.4%), whereas Lf was superior to the other markers in UC (83.3%). The comprehensive activity index yielded a further improvement of sensitivity and specificity, with a diagnostic accuracy of 95.3% for UC patients. CONCLUSION: The fecal markers Lf, Cal, and PMN-e are able to differentiate active IBD from inactive IBD as well as from IBS. None of these three stool markers is consistently superior in its ability to reflect endoscopic inflammation, but all three are superior to CRP in their diagnostic accuracy. A combination of the stool markers with the CRP and a disease-specific activity index in a categorical comprehensive activity index can increase the diagnostic accuracy with reference to the endoscopic inflammation in UC.

[Assessment of severity of acute pancreatitis in early stage].

OBJECTIVE: To assess the accuracy of indexes for predicting severe acute pancreatitis (SAP). METHODS: Thirty-nine patients suffering from acute pancreatitis (AP) were randomly selected, including 20 SAP cases and 19 mild acute pancreatitis (MAP) cases. The levels of polymorphonuclear leucocyte-elastase (PMN-E), serum phospholipase A2 (PLA2), pancreatic PLA2 (Pan-PLA2), PLA2 catalytic activity (CA-PLA2), amylase, as well as C-reactive protein (CRP) were determined Acute Physiology and chronic health evaluation II (APACHE II) was scored in every patient. Sensitivity and specificity of all the parameters were assessed, and receiver operator characteristic curve (ROC) was plotted. Positive predictive value, negative predictive value, and overall accuracy were then analyzed. RESULTS: PMN-E, CRP and CA-PLA2 were obviously higher in SAP than in MAP, and were indicative of the severity of the disease (all P < 0.01). Pan-PLA2 and amylase of AP patients raised at the onset of the disease, and they showed no difference between the SAP groups and MAP groups. When SAP was predicted by PMN-E, sensitivity was 94.5%, specificity was 99.4%, positive predictive value was 97.8%, negative predictive value was 99.4%, overall accuracy was 98.7%, higher than other indexes. When SAP was Predicted by CRP, the overall accuracy was also high and reached 84.0%. CONCLUSION: PMN-E, CA-PLA2, CRP, and APACHE II are all indexes for the diagnosis of SAP. PMN-E is found to be the best index in predicting SAP.

[Recent advances in biochemical diagnosis and assessment of severity in acute pancreatitis].

Serum amylase is most commonly used as a biochemical marker of acute pancreatitis (AP). But it lacks specificity. The serum lipase level is more accurate and a better marker. Serum elastase -1 level is specific and remains elevated longer, but its radioimmunoassay is not routinely used. Recently, it can be rapidly measured by latex turbidometric immunoassay with automatic analyzer. Biochemically, only CRP test is available and useful to assess severity, but its sensitivity is unacceptably low in the early course of the disease. Urinary trypsinogen activation peptide (TAP) or trypsinogen-2 is an earlier marker. Increasing knowledge of the inflammatory process in AP has led to possibly useful biochemical indicators of severity, such as cytokines, nonpancreatic synovial type group II PLA2 or granulocyte elastase.

Quantitative assessment of apoptotic and proliferative gingival keratinocytes in oral and sulcular epithelium in patients with gingivitis and periodontitis.

BACKGROUND: Periodontal disease is caused by a chronic infection inducing an inflammatory reaction that leads to a breakdown of tooth-supporting tissue. The maintenance of an equilibrium between the host defence and microorganisms in the sulcus is essential to preserve health. All multicellular organisms have mechanisms for killing their own cells, and use physiological cell death for defence, development, homeostasis and ageing. Apoptosis and proliferation are very important phenomena in regulating this and a disturbance is often associated with disease e.g. cancer, AIDS, Alzheimer's disease, rheumatoid arthritis. OBJECTIVE: The aim of this study was to determine whether the number of apoptotic and proliferative gingival keratinocytes differed between patients with gingivitis and those with periodontitis. MATERIAL AND METHODS: The distribution of neutrophil elastase, PCNA/cyclin, DNA fragmentation (apoptosis) and p53 was determined with immunocytochemical techniques. We used paraffin-embedded sections from gingival biopsies and did quantitative analyses. RESULTS AND CONCLUSION: These showed that 5-12% of the keratinocytes in the basal layers of the epithelium proliferated in the two groups. Fewer apoptotic cells were seen in the oral epithelium than in the sulcus in all subjects in both groups. Only in the most apical part of the sulcus, close to the junctional epithelium, did the number of apoptotic keratinocytes exceed the proliferative ones in patients with periodontitis.

Nasal lavage as a tool for the assessment of upper-airway inflammation in adults and children.

The prevalence of respiratory allergies has increased over the last 20 years, highlighting the need for a simple and noninvasive tool to investigate, in a clinical and epidemiological context, airway-inflammation mechanisms encountered in allergic and inflammatory processes. The nose, as the first region of the respiratory tract to come in contact with airborne pollutants, is easily explored with the use of nasal lavage (NL). We evaluated an NL method for adults and children, along with its reproducibility and capacity to separate different subgroups. NL reproducibility, assessed in 10 healthy, nonsmoking adults on three different occasions, was determined with the use of the intraclass coefficient of correlation for such inflammatory markers as total cell count, albumin, urea, neutrophil elastase, alpha(1)-antitrypsin, interleukin-6, and interleukin-8. Using this NL method, we analyzed nasal markers of 50 healthy adults (smokers and nonsmokers) and 12 healthy children. Our NL method demonstrated high reproducibility with regard to total cell count, albumin, urea, and alpha(1)-antitrypsin (intraclass correlation coefficient > 0.75). Compared with NL results in nonsmokers, NL in heavy smokers revealed significant increased concentrations of total cell counts and interleukin-8 and significant decreased concentrations of interleukin-6. These findings suggest that NL can be used as a tool in the assessment of inflammation because it has the correct reproducibility and can discriminate between heavy smokers and nonsmokers. Moreover, the use of this standardized method in children is feasible.

Assessment by flow cytometry of peripheral blood leukocyte enzymatic activities in HIV patients.

Leukocyte enzymatic activities are important in non-specific protection against bacterial infections, but traditional methods for the detection of intracellular enzymatic activities rely on cumbersome and complex assays. The development of specific substrates, which become fluorescent upon degradation of the biomolecule after its passive entry into intact cells, permits a simplified evaluation of leukocyte enzymatic activities. We have used this method to assess intracellular elastase, collagenase and cathepsin D activities of peripheral blood leukocytes using flow cytometry in a series of HIV patients and healthy controls. Monocytes displayed the highest enzymatic activities for the three proteases tested. In HIV-infected patients, the collagenase and cathepsin D activities of monocytes were significantly lower, whereas the elastase and cathepsin D activities of polymorphonuclear cells were elevated. Slightly higher elastase activity was detected in the lymphocytes of patients. This study demonstrates the feasibility of this new method for the study of intracytoplasmic enzymatic activities. Significant variations were observed in the peripheral blood of HIV-infected patients and different patterns were especially evident in monocytes and polymorphonuclear cells.