The Bradyrhizobium Sp. LmicA16 Type VI Secretion System Is Required for Efficient Nodulation of Lupinus Spp.

Many bacteria of the genus Bradyrhizobium are capable of inducing nodules in legumes. In this work, the importance of a type VI secretion system (T6SS) in a symbiotic strain of the genus Bradyrhizobium is described. T6SS of Bradyrhizobium sp. LmicA16 (A16) is necessary for efficient nodulation with Lupinus micranthus and Lupinus angustifolius. A mutant in the gene vgrG, coding for a component of the T6SS nanostructure, induced less nodules and smaller plants than the wild-type (wt) strain and was less competitive when co-inoculated with the wt strain. A16 T6SS genes are organized in a 26-kb DNA region in two divergent gene clusters of nine genes each. One of these genes codes for a protein (Tsb1) of unknown function but containing a methyltransferase domain. A tsb1 mutant showed an intermediate symbiotic phenotype regarding vgrG mutant and higher mucoidity than the wt strain in free-living conditions. T6SS promoter fusions to the lacZ reporter indicate expression in nodules but not in free-living cells grown in different media and conditions. The analysis of nodule structure revealed that the level of nodule colonization was significantly reduced in the mutants with respect to the wt strain.

Phenolic and tannin compounds in subtropical shrubs (Bituminaria bituminosa, Chamaecytisus proliferus, and Adenocarpus foliosus) and the effects on in vitro digestibility.

The objective of this study was to contribute to the characterization of nutritional potentials, with special attention to the total phenol and condensed tannins content and the effects on in vitro digestibility of some browsing shrub legumes traditionally used for ruminant feeding. The varieties selected were Bituminaria bituminosa var. bituminosa (BBB), Bituminaria bituminosa var. albormaginata (BBA), Chamaecytisus proliferus var. palmensis (CPP), Chamaecytisus proliferus var. canariae (CPC), and Adenocarpus foliosus (AF). Chemical composition, in vitro digestibility, and net energy for lactation (NE) were analyzed and compared among varieties; the effects of total phenol (TP) and condensed tannins (CT) compounds on digestibility were estimated. Condensed tannins ranged from 1 to 5 g/kg DM of tannins, while total phenols ranged from 9 to 32 g/kg DM of tannins. In vitro organic matter digestibility (OMD) ranged from 0.52 to 0.71, and estimated net energy for lactation (NE) ranged from 4.2 to 5.8 MJ/kg DM, with significant differences among all the varieties. This Chamaecytisus varieties (CPC, CPP) have higher feeding values, following CPC > CPP > AF > BBB > BBA in order. Although there were condensed tannins and total phenols in all the shrubs analyzed, total phenol contents differed significantly between the shrubs. The effect of the content of the total phenols and of the tannins condensed in the OMD of the varieties of shrubs analyzed, shows that the higher amounts of total phenols in some of the varieties of shrubs analyzed, increased the in vitro digestibility (OMD) of the shrubs but high amounts of CT decreased OMD.

Effects of replacing rye-grass (Lolium spp.) hay by banana (Musa acuminata L.) by-products on feed intake, growth, and feed conversion rate of Canary hair sheep breed (Pelibuey) lambs.

The objective of this study was to evaluate the effects of replacing rye-grass (Lolium spp.) hay with banana (Musa acuminata L.) by-products on feed intake, average daily gain (ADG), and feed conversion rate (FCR) in Pelibuey (hair sheep breed) lambs. The lambs (n = 22; initial BW = 14.8 ± 2.5 kg) were individually housed and fed on two different types of diet for 58 days: the first group (experimental diet) received as forage a mixture of fresh banana by-products composed of leaves and pseudostem. The second group received commercial rye-grass hay (conventional diet) as a fiber source. Both groups received a supplementary commercial concentrate food. The total daily forage intake and FCR were lower (P < 0.001) in animals fed on the conventional diet (rye-grass hay) than animals fed on the experimental diet (banana by-products). However, there were no significant differences in total dry matter intake, total digestible energy intake, total body weight gain, and ADG between the two groups. The results obtained in this study suggest that banana by-products could be used as alternative forage for Pelibuey lambs raised in subtropical conditions.

Emotion-regulation ability, role stress and teachers' mental health.

BACKGROUND: Work-related stressors, including role ambiguity and role conflict, are related to psychological maladjustment and mental ill-health. However, to date, the role of personal resources such as emotion-regulation ability (ERA) in the prediction of mental health indicators has not been addressed. AIMS: To examine whether ERA would contribute to explaining teachers' depression, anxiety and stress symptoms beyond role ambiguity and role conflict. METHODS: We carried out a cross-sectional questionnaire survey. We used a correlation matrix and hierarchical regression models to analyse the data. RESULTS: Three hundred and thirty-six Spanish teachers (185 female) from several grade levels completed the surveys (40% response rate). Role ambiguity and role conflict were positively related to depression, anxiety and stress symptoms. ERA was negatively related to teachers' scores on depressive, anxious and stress symptoms, with predictive power above the main effects of role ambiguity and role conflict. An interaction between role ambiguity and ERA was also significant in predicting depression. CONCLUSIONS: Our study provides preliminary evidence suggesting the development of integrative models considering work-related stressors along with personal resources such as ERA aiming to prevent teachers' mental ill-health. Future studies should examine the influence of ERA on psychological symptoms using longitudinal designs.

[Therapeutic restraint management in Intensive Care Units: Phenomenological approach to nursing reality]. / Manejo de la inmovilización terapéutica en Unidades de Cuidados Críticos: aproximación fenomenológica a la realidad enfermera.

AIMS: To identify nursing experience on physical restraint management in Critical Care Units. To analyse similarities and differences in nursing experience on physical restraint management according to the clinical context that they are involved in. METHOD: A multicentre phenomenological study was carried out including 14 Critical Care Units in Madrid, classified according to physical restraint use: Common/systematic use, lacking/personalised use, and mixed use. Five focus groups (23 participants were selected following purposeful sampling) were convened, concluding in data saturation. Data analysis was focused on thematic content analysis following Colaizzi's method. FINDINGS: Six main themes: Physical restraint meaning in Critical Care Units, safety (self-retreat vital devices), contribution factors, feelings, alternatives, and pending issues. Although some themes are common to the 3 Critical Care Unit types, discourse differences are found as regards to indication, feelings, systematic use of pain and sedation measurement tools. CONCLUSIONS: In order to achieve real physical restraint reduction in Critical Care Units, it is necessary to have a deep understanding of restraints use in the specific clinical context. As self-retreat vital devices emerge as central concept, some interventions proposed in other settings could not be effective, requiring alternatives for critical care patients. Discourse variations laid out in the different Critical Care Unit types could highlight key items that determine the use and different attitudes towards physical restraint.

Social support from health care providers is associated with reduced illness intrusiveness in hemodialysis patients.

BACKGROUND: End-stage renal disease (ESRD) disrupts patients' life styles, interests and activities negatively affecting their quality of life. Social support has been previously associated with favorable health outcomes. However, no study has examined the association of social support from health care providers with perceived health and ESRD intrusiveness on patients' lives. METHODS: A self-administered questionnaire was completed by 1,238 Italian hemodialysis patients. The Self-Rated Health (SRH) and the Illness Intrusiveness Rating Scale (IIRS) assessed disease burden. 10 items assessed social support from health care providers (SS-HC). The nursing staff of each center provided patients' clinical information. Linear regression was used to assess correlates of SRH and IIRS. Mediational analysis was used to assess direct and indirect associations of SS-HC with SRH through IIRS. RESULTS: Higher SS-HC was associated with smaller IIRS and higher SRH. Further correlates of better SRH were younger age, no post-dialysis hypotension, no diabetes and cardiovascular diseases, better sleep quality, and smaller burden of oral therapy. CONCLUSIONS: Our results suggest that social support might reduce illness burden and improve patients' perceived health. Further research should assess the efficacy and cost-effectiveness of structured support programs for dialysis patients.

Epigenetic control of the EWS­FLI1 promoter in Ewing's sarcoma.

Ewing sarcoma (ES) is a primary bone marrow tumor that very rarely develops in extra­osseous tissues, such as lung. The hallmark of ES tumors is a translocation between chromosomes 11 and 22, resulting in a fusion protein, commonly referred to as EWS­FLI1. The epigenetic profile (histone acetylation and methylation enrichment of the promoter region) that may regulate the expression of the aberrant transcription factor EWS­FLI1, remains poorly studied and understood. Knowledge of epigenetic patterns associated with covalent histone modification and expression of enzymes associated with this process, can contribute to the understanding of the molecular basis of the disease, as well as to the identification of possible molecular targets involved in expression of the EWS­FLI1 gene, so that therapeutic strategies may be improved in the future. In the present study, the transcriptional activation and repression of the EWS­FLI1 fusion gene in ES was accompanied by selective deposition of histone markers on its promoter. The EWS­FLI1 fusion gene was evaluated in two patients with ES using conventional cytogenetic, fluorescence in situ hybridization and nested PCR assays, which revealed that the aberrant expression of the EWS­FLI1 gene is accompanied by enrichment of H3K4Me3, H3K9ac and H3K27ac at the promoter region.

Reflections arising from the COVID-19 pandemic. / Reflexiones derivadas de la pandemia COVID-19.

While we were drafting the recommendations for the joint contingency plan between the Spanish Society of Intensive Care and Coronary Unit Nursing (SEEIUC) and the Spanish Society of Intensive and Critical Care Medicine and Coronary Units (SEMICYUC), predictions were overtaken by events with regard to the magnitude of the COVID-19 pandemic. Members informed us of the lack of personal protection equipment (PPE), the rapid provision of improvised ICUs in various hospital areas and the recruitment of nurses to cover shifts. The failure to recognise the specialty of critical care nursing, included in the macro-specialty of medical-surgical nursing and not yet developed, has highlighted the urgent need to learn from our mistakes: specialisation, increase the number of nurses in teams and protect the public health system.

Physical restraint in critical care units from the experience of doctors and nursing assistants: In search of an interdisciplinary interpretation. / La contención mecánica en unidades de cuidados críticos desde la experiencia de los médicos y técnicos en cuidados auxiliares de enfermería: buscando una lectura interdisciplinar.

OBJECTIVES: The study aim was to explore the experience of doctors and nursing assistants in the management of physical restraint (PR) in critical care units. METHOD: A multicentre phenomenological study that included 14 critical care units (CCU) in Madrid (Spain). The CCU were stratified according to their use of physical restraint: "frequently used" versus "seldom used". Three focus groups were formed: the first comprised nursing assistants from CCUs that frequently used physical restraint, the second comprised nursing assistants from CCUs that seldom used physical constraint, and the final group comprised doctors from both CCU subtypes. Sampling method: purposive. DATA ANALYSIS: thematic content analysis. Data saturation was achieved. RESULTS: Four principle themes emerged: 1) concept of safety and risk (patient safety versus the safety of the professional), 2) types of restraint, 3) professional responsibilities (prescription, recording, and professional roles) and 4) "zero restraint" paradigm. The conceptualisation regarding the use of physical contentions shows differences in some of the principal themes, depending on the type of CCU, in terms of policies, use and management of physical constraint (frequently used versus seldom used). CONCLUSIONS: The real reduction in the use of physical restraint in CCU must be based on one crucial point: acceptance of the complexity of the phenomenon. The use of physical restraint observed in the different CCU is influenced by individual, group and organisational factors. These factors will determine how doctors and nursing assistants interpret safety and risk, the centre of care (patient or professional-centred care), the concept of restraint, professional responsibilities and interventions, interactions of the team and the leadership.

[Contingency plan for the intensive care services for the COVID-19 pandemic]. / Plan de contingencia para los servicios de medicina intensiva frente a la pandemia COVID-19.

In January 2020, the Chinese authorities identified a new virus of the Coronaviridae family as the cause of several cases of pneumonia of unknown aetiology. The outbreak was initially confined to Wuhan City, but then spread outside Chinese borders. On 31 January 2020, the first case was declared in Spain. On 11 March 2020, The World Health Organization (WHO) declared the coronavirus outbreak a pandemic. On 16 March 2020, there were 139 countries affected. In this situation, the Scientific Societies SEMICYUC and SEEIUC have decided to draw up this Contingency Plan to guide the response of the Intensive Care Services. The objectives of this plan are to estimate the magnitude of the problem and identify the necessary human and material resources. This is to provide the Spanish Intensive Medicine Services with a tool to programme optimal response strategies.

Novel association of the CD226 (DNAM-1) Gly307Ser polymorphism in Wegener's granulomatosis and confirmation for multiple sclerosis in German patients.

Recently, there has been increasing evidence that a non-synonymous exchange (Gly307Ser) in the gene for CD226 is linked to several autoimmune diseases including type 1 diabetes, multiple sclerosis (MS), rheumatoid arthritis and Grave's disease. Here we present evidence that this polymorphism also predisposes to Wegener's granulomatosis (WG), an autoimmune condition belonging to the group of ANCA (antineutrophil cytoplasmic autoantibody)-associated vasculitides. We found a significant association of the 307Ser allele in separate panels of 520 Northern German (P=0.016, odds ratio (OR)=1.20) and 122 Southern German (P=0.020, OR=1.37) WG cases compared with 1226 healthy controls. The importance of this single-nucleotide polymorphism in the etiopathology of ANCA-associated vasculitides is supported by similar effect sizes that we found in British WG cases (n=105) and German patients with Churg-Strauss syndrome (n=119), which, however, miss significance level because of the relatively small cohorts available for these rare disorders. Finally, we confirm the association with MS in a cohort of 422 German patients (P=0.011, OR=1.23).

[Pathogenesis and treatment of vascular calcification in CKD]. / Invecchiamento cardiovascolare nel soggetto uremico: nuove acquisizioni.

Increased vascular calcification is a major cause of cardiovascular events in patients with chronic kidney disease (CKD). It is the result of an active ossification process counteracted by ''bone'' proteins such as osteopontin, alkaline phosphatase, osteoprotegerin, and osteocalcin. Chronic kidney disease - mineral and bone disorder (CKD-MBD) is a systemic disorder of mineral and bone metabolism that occurs in CKD. In addition to abnormalities in the serum calcium and phosphate profile, CKD-MBD is characterized by abnormalities of bone turnover, mineralization, volume and growth as well as vascular calcification. Considering that the presence and extent of vascular calcification in CKD portend a poor prognosis, many efforts have been made to shed light on this complicated phenomenon to prevent vascular calcium deposition and its progression. Indeed, careful control of calcium load, serum phosphate and parathyroid hormone along with the use of calcium-free phosphate binders and vitamin D receptor activators represent a new therapeutic armamentarium to improve quality of life and reduce mortality in CKD.

Targeting expression to megakaryocytes and platelets by lineage-specific lentiviral vectors.

Essentials Platelet phenotypes can be modified by lentiviral transduction of hematopoietic stem cells. Megakaryocyte-specific lentiviral vectors were tested in vitro and in vivo for restricted expression. The glycoprotein 6 vector expressed almost exclusively in megakaryocytes. The platelet factor 4 vector was the strongest but with activity in hematopoietic stem cells. SUMMARY: Background Lentiviral transduction and transplantation of hematopoietic stem cells (HSCs) can be utilized to modify the phenotype of megakaryocytes and platelets. As the genetic modification in HSCs is transmitted onto all hematopoietic progenies, transgene expression from the vector should be restricted to megakaryocytes to avoid un-physiologic effects by ectopic transgene expression. This can be achieved by lentiviral vectors that control expression by lineage-specific promoters. Methods In this study, we introduced promoters of megakaryocyte/platelet-specific genes, namely human glycoprotein 6 (hGP6) and hGP9, into third generation lentiviral vectors and analyzed their functionality in vitro and in vivo in bone marrow transplantation assays. Their specificity and efficiency of expression was compared with lentiviral vectors utilizing the promoters of murine platelet factor 4 (mPf4) and hGP1BA, both with strong activity in megakaryocytes (MKs) used in earlier studies, and the ubiquitously expressing phosphoglycerate kinase (hPGK) and spleen focus forming virus (SFFV) enhancer/promoters. Results Expression from the mPf4 vector in MKs and platelets was the strongest similar to expression from the viral SFFV promoter, however, the mPf4 vector, also exhibited considerable off-target expression in hematopoietic stem and progenitor cells. In contrast, the newly generated hGP6 vector was highly specific to megakaryocytes and platelets. The specificity was also retained when reducing the promoter size to 350 bp, making it a valuable new tool for lentiviral expression in MKs/platelets. Conclusion MK-specific vectors express preferentially in the megakaryocyte lineage. These vectors can be applied to develop murine models to study megakaryocyte and platelet function, or for gene therapy targeting proteins to platelets.

Nucleotide sequence and organization of an H2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames.

The nucleotide sequence of a 3.2 kb region following the hydrogenase structural operon (hupSLCDEF) in the H2-uptake gene cluster from Rhizobium leguminosarum by viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16.3 (HupG), 30.5 (HupH), 8.0 (HupI), 18.4 (HupJ) and 38.7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHIJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.

Duvernoy's gland secretion of Philodryas patagoniensis from the northeast of Argentina: its effects on blood coagulation.

Duvernoy's gland secretion of Philodryas patagoniensis exhibits high hemorrhagic activity, containing enzymes that are able to degrade the vascular wall. In this work we aim to determine if the secretion can also affect the hemostatic system by causing changes in blood coagulation. Procoagulant and coagulant activities were evaluated on plasma and fibrinogen, respectively. The delay in the thrombin clotting time of fibrinogen previously incubated with the secretion was also determined. Specific hydrolysis of fibrinogen and fibrin incubated with the secretion at different time intervals was shown by electrophoresis on polyacrylamide gel. To determine the structural characteristics of the enzymes degrading fibrinogen and fibrin, secretion were incubated in the presence of 45 mM Na(2)EDTA, 40 mM Benzamidine, and/or 2 mM PMSF before the incubation with fibrinogen or fibrin, respectively. The effect in vivo was investigated in adult male rats injected with different dose of secretion, aliquots of blood were withdrawn at different time intervals, and the fibrinogen concentration was determined. Duvernoy's gland secretion of P. patagoniensis did not clot plasma or fibrinogen. It exhibited a potent fibrinogenolytic activity degrading the Aalpha-chain faster than the Bbeta-chain, whereas gamma-chain was resistant. This latter corresponded with a strong delay in the thrombin clotting time of fibrinogen (4 mg/ml) pre-incubated with the secretion, being 9.53 microg the amount of protein from Duvernoy's gland secretion that increased the thrombin clotting time from 20 to 60 s. In vivo, the loss of rat plasma fibrinogen was proportional to the amount of secretion injected. The secretion also hydrolyzed fibrin degrading the alpha-monomer. Inhibition studies with Na(2)EDTA, Benzamidine, and/or PMSF showed that metalloproteinases and serinoproteinases are the main enzymes responsible for the hydrolyzing activity on fibrinogen and fibrin. All these results demonstrate that Duvernoy's gland secretion of P. patagoniensis possesses enzymes able to hydrolyze plasma components playing a relevant role in the blood coagulation. These hydrolyzing activities and those acting on the wall of blood vessels let the secretion exhibit a high hemorrhagic activity, which may result in permanent sequelae or even cause the death of the victims bitten by this colubrid snake.

Facial changes in adult uremic patients on chronic dialysis: possible role of hyperparathyroidism.

BACKGROUND: Uremic patients on regular dialytic treatment (RDT) are often affected by a complex metabolic syndrome leading to osteodystrophy. Bone changes are primarily due to high bone turnover, often combined with a mineralization defect leading to increased bone fractures and bone deformities. Although rarely considered, the craniofacial skeleton represents one of the peculiar targets of this complex metabolic disease whose more dramatic pattern is a form of leontiasis ossea. This complication, although described, has never been evaluated in depth nor quantitatively assessed. In order to assess facial deformities in uremic conditions and to understand the possible relation with hyperparathyroidism, we undertook a quantitative evaluation of soft facial structures in a cohort of uremic patients undergoing RDT. METHODS: The three-dimensional coordinates of 50 soft-tissue facial landmarks were obtained by an electromagnetic digitizer in 10 male and 10 female patients with chronic renal insufficiency aged 53-81 years, and in 34 healthy individuals of the same age, ethnicity and sex. Uremic patients were enrolled according to hyperparathyroid status (PTH < 300 pg/mL and PTH > 500 pg/mL). From the landmarks, facial distances, angles and volumes were calculated according to a geometrical face model. RESULTS: Overall, the uremic patients had significantly larger facial volumes than the reference subjects. The effect was particularly evident in the facial middle third (maxilla), leading to an inversion of the mandibular-maxillary ratio. Facial dimensions were increased in all three spatial directions: width (skull base, mandible, nose), length (nose, mandible), and depth (mid face, mandible). The larger maxilla was accompanied by a tendency to more prominent lips (reduced interlabial angle). Some of the facial modifications (nose, lips, mandible) were significantly related to the clinical characteristics of the patients (age, duration of renal insufficiency and PTH levels). CONCLUSIONS: This report, the first in the literature, shows that facial structures of uremic patients are enlarged in comparison with matched normal subjects and that increased bone turnover could be responsible--at least in part--for facial bone changes.

[New insights in the pathogenesis of secondary hyperparathyroidism]. / Nuove acquisizioni nella patogenesi dell'iperparatiroidismo secondario.

Parathyroid gland growth is a major cause of secondary hyperparathyroidism in renal failure. It is well known that high serum phosphate levels, low serum calcium levels and vitamin D deficiency are the three promoters of parathyroid hyperplasia in renal failure. Recent studies have investigated in depth the potential role of growth factors (transforming growth factor alpha) and their receptors (epidermal growth factor receptor) in the pathogenesis of parathyroid cell hyperplasia in chronic renal failure. The identification of molecular mechanisms involved in calcium, phosphate and vitamin D manipulations in an experimental renal failure model could help design more effective therapy for secondary hyperparathyroidism in uremic patients.

Thromboxane augmentation of alloreactive T cell function.

Thromboxane (Tx) plays a vital role in the dysfunction and ultimate rejection of MHC-disparate renal allografts. In addition to its potent vasoconstrictory properties, in vivo studies have implied that Tx is capable of promoting immune cytotoxic T cell function within transplants. In this study, we have examined the in vitro effect of Tx inhibition on alloreactive immune cells using MHC-disparate mouse strain combinations. Coculture of either Tx-synthetase or Tx-receptor inhibitors modified the response of unprimed mouse lymphoid populations in a primary MLR, implying that Tx inhibition and not endoperoxide shunting was responsible for the modulatory effects seen. For example, B10.S lymphoid cells displayed decreased proliferation to H-2 disparate B10.A cells with Tx inhibitors present during the MLR, at pharmacologically active drug concentrations. Moreover, in vitro addition of TxA2 had an augmentory effect on the response in the primary and secondary MLR. Interleukin 2 production and percentages of T cell populations in the primary MLR were not affected by the presence of these compounds, although CD4 and CD8 expression was often increased in the treated populations. Finally, alloreactive primed effector cells also displayed reduced proliferation to specific alloantigen in a secondary MLR when Tx inhibitors were also present, although responses to IL-2 by T cells were not influenced by thromboxane inhibition. These data imply that thromboxane is an important immunoregulatory mediator capable of potentiating the function of naive and primed alloreactive immune T cell populations crucial to the rejection of the transplant.

Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer's Disease (AD) individuals.

In order to investigate the possibility of whether or not the lymphocytes of patients with Alzheimer's Disease (AD) are in an activated state, blood mononuclear cells from 45 AD patients and 45 healthy age matched controls were immunophenotyped by measuring the expression of CD3, CD4, CD7, CD8, CD25, CD28, CD56 and HLA-DR by flow cytometry. Circulating and in-vitro-produced cytokines were also measured by ELISA tests. CD7 and CD8 were significantly decreased in AD patients (48.3% and 18.2%, respectively) when compared to healthy subjects (63.2% and 28.3%, respectively). A significant increase in the CD4, CD25 and CD28 antigen expression was also observed in the AD group (55.3% 24.8% and 65.1%) with respect to healthy subjects (44.5%, 10.3% and 54.3%). In addition there was a significant difference in the extent of apoptosis in lymphocyte culture, as measured by mean fluorescence intensity (MFI) of Fas antigen (CD95) expression on CD4+ T cells in 6 AD patients (MFI = 36% and 43%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively) with respect to 6 healthy individuals (MFI = 24% and 31%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively), as well as in T-cell proliferation assay. A decline of Fas antigen expression on CD8+ subset was observed in the AD group with both stimuli (19% and 28%) comparing to the control group (29% and 39%). No differences were observed on circulating cytokines and spontaneous in vitro production of proinflammatory interleukin 1beta (IL-1beta), Tumor Necrosis Factor-alpha (TNF-alpha), IL-6 and IL-10 cytokines. Lipopolysaccharide (LPS)-stimulated in vitro production of IL-1beta, TNF-alpha, IL-6 and IL-10 measured by a whole blood culture system was significantly higher in AD patients comparing to controls. Furthermore, the observed differences were more evident at late stages of disease. These findings suggest that immunological tests, based on lymphocyte immunophenotyping combined with pro-inflammatory cytokine determinations and measurement of apoptosis in peripheral blood might represent a useful tool to obtain more insight into the pathogenesis of AD and into the level of immune activation which could characterize the pathological state of lymphocytes from individual AD patients.

Immunopathological characteristics of in situ T-cell subpopulations in human immunodeficiency virus-associated nephropathy.

Human immunodeficiency virus-associated nephropathy (HI-VAN) is a common form of nephropathy present in HIV-infected individuals that clinically presents with proteinuria that is frequently in the nephrotic range, less often with hematuria, and with a course that may evolve to irreversible azotemia ultimately resulting in renal failure. Pediatric and adult HIV-positive patients both experience HIVAN morphologically after displaying focal segmental glomerulosclerosis, diffuse mesangial hyperplasia, microcystic tubular dilatation, interstitial inflammation, edema, and fibrosis. There is minimal information regarding the interstitial inflammatory cell infiltrate, despite the possibility that these cells may play an important role in the etiology of HIVAN. This study was designed to characterize and compare several morphological and immunopathological features of clearly established HIVAN, particularly the hematopoietic cell markers present on the interstitial inflammatory cells and the state of T-lymphocyte activation (ie, class II expression). Quantitative grading of HIVAN kidneys showed that CD4-positive and CD8-positive T cells comprised the major cell populations in the interstitium, often with CD4-positive T cells exceeding or being equivalent in number to CD8-positive T cells. B cells and macrophages were negligible components of the infiltrate. Human leukocyte antigen-DR class II molecules were found to be increased on the interstitial T cells as well as on all glomerular cells and endothelial cells. There was no significant relationship established between the immunophenotype of the interstitial inflammatory cells and other morphological, ultrastructural, immunofluorescent, or clinical features. These data imply that the inflammatory infiltrate in HIVAN is largely composed of activated T cells. At this point the role of these interstitial T cells in HIVAN is undetermined, although it can be speculated that they may be participating as antiviral or autoreactive immune effector cells imparting renal injury in this entity.