Direct Immunofluorescence Technique Research Articles

Acanthamoeba spp. as vehicle and reservoir of adenoviruses

Adenoviruses are important pathogens which are responsible for human enteritic, respiratory and eye infections. These viruses have been found to be prevalent in several natural and artificial water reservoirs worldwide. Free-living amoebae (FLA) have been recovered from similar water reservoirs, and it has been shown that FLA may act as reservoirs or vehicles of various microorganisms living in the same environment. To examine the ability of FLA to harbour adenoviruses, an in vitro study was conducted. Several Acanthamoeba strains were ‘co-cultivated’ with adenoviruses (adenoviruses 11 and 41), grown on A549 cells, using a proven test protocol. After phagocytosis and ingestion, the adenoviruses could be found within the cytoplasm of the Acanthamoeba trophozoites. The intake of the viruses into the cytoplasm of the trophozoites was demonstrated in an Acanthamoeba castellanii strain with the help of fluorescence microscopy and electron microscopy. An adenovirus DFA kit, which utilizes a direct immunofluorescent antibody technique for identifying adenovirus in infected tissue cultures, was used. In our study, it was demonstrated that adenoviruses were incorporated into the host amoebae (Acanthamoeba sp. Grp. II, three strains). So far, there were only a few publications concerning the relationship of free-living amoebae and viruses; only one of these described the detection of adenoviruses within acanthamoebae with molecular biological methods. We conducted this descriptive study to further examine the association between viable adenoviruses and FLA. To our knowledge, this is the first study to demonstrate directly the adenoviruses within FLA as vectors and vehicles. Therefore, we concluded that free-living amoebae appear able to act as carriers or vectors of the adenoviruses and thus may play a certain role in the dispersal of adenoviruses.

Mixed phenotype acute leukemia of T/myeloid type with a prominent cellular heterogeneity and unique karyotypic aberration 45,XY, dic(11;17)

A 26-yr-old male patient with mixed phenotype acute leukemia of T/myeloid type with prominent leukemic cell heterogeneity, and the presence of a so far unreported karyotype aberration in this type of acute leukemia 45,XY, dic(11;17)(11qter→11p11.2::17p11.2→17qter) is presented. Flow immunocytometry was performed by direct multicolor immunofluorescent technique on bone marrow aspirates. Cytogenetic analyses were performed using G-banding method by direct preparation of unstimulated bone marrow cells and following 24 hours of culture in RPMI 1540 culture medium with 25% fetal calf serum at 37°C RESULTS: The flow immunocytometry of bone marrow nucleated cells revealed the existance of three distinct blast cell populations with overlapping immunophenotypes. Predominant blast cell population had an early myeloid phenotype and aberrant expression of CD7 antigen (HLA-DR(+), CD34(+), anti-MPO(+), CD117(+), CD33(+), CD13(+), CD7(+low), cyCD3(-), TdT(-)). The other two blast cell populations, smaller in cell diameter and less sizable in cell proportion, both shared the T-lymphoid features. The patient was treated with ADE protocol (etoposide, cytarabine and doxorubicine). A complete remission was achieved and lasted 5 months. A case of MPAL with complex biological features, 45,XY, dic(11;17)(11qter→11p11.2::17p11.2→17qter) karyotype and an aggressive, therapy-resistant clinical course, is presented.

Quantification of Feto-Maternal Hemorrhage: Selection of Techniques for a Resource-Poor Setting

Background: Though RhD sensitization in RhD-negative mothers is now almost eradicated in the developed world, it continues to be a major health problem in developing nations like India. Inadequate immunoprophylaxis is the main reason. Adequate dose calculation of anti-D Ig is possible through estimation of correct feto-maternal hemorrhage (FMH) volume. In this regard, different methods have been used. Methods: We evaluated three quantitative techniques of estimating FMH: the Kleihauer-Betke test (KBT) and two flow cytometry (FC) techniques, i.e., the indirect immunofluorescence technique (IIFT) and direct immunofluorescence technique (DIFT). Stock solutions of both RhD-positive and D-negative cells were made, and 7 serial dilutions of RhD-positive cord cells in D-negative adult cells were prepared. Result: Both KBT and FC approximated the expected concentration of fetal RhD-positive cells in all mixtures tested. In both methods, an underestimation of fetal RhD-positive cells was observed when their expected concentration was ≧0.75%. Conclusion: Though FC is the most sensitive of all techniques, very few laboratories in developing nations can afford such a costly device, so it will be prudent for them to use KBT as the gold standard due to its rapidity, simplicity and affordability.

Multiparameter Flow Cytometry Analysis of Peripheral Blood T, NK and Dendritic Cells From High-Risk Smoldering Multiple Myeloma Patients Treated with Lenalidomide and Dexamethasone

AbstractAbstract 1906Lenalidomide is an immunomodulatory agent that interacts with different components of the immune system by altering cytokine production, regulating T cells and increasing NK cell cytotoxicity. In multiple myeloma (MM), lenalidomide is approved for use in combination with dexamethasone in patients who have received at least one prior therapy. Recent observations have shown that dexamethasone enhances the anti-myeloma effect of lenalidomide; however, dexamethasone may also antagonize the immunomodulatory properties of lenalidomide.In the present study we evaluated by multiparameter flow cytometry (MFC) peripheral blood (PB) T, NK and dendritic (plasmacytoid, myeloid and monocytic) cells (DC) from high-risk smoldering MM (SMM) patients, defined by the presence of at least 2 of the 3 following criteria at diagnosis: bone marrow plasma cell (BMPC) infiltration ≥10%; and/or high M-component (IgG≥30g/L or IgA≥20g/L or B-J Protein>10g/L); and/or ≥95% myelomatous-PC/BMPC and immune paresis. SMM patients were treated according to the QuiReDex trial (NCT 00480363): an induction phase of nine four-week cycles of lenalidomide plus dexamethasone (LenDex) followed by maintenance with lenalidomide until disease progression. In this ongoing study, immunophenotypic data is available in 53 patients at diagnosis (baseline), 30 after 3 cycles of LenDex and 22 at the end of induction therapy (9th cycle). Here we will focus on the 22 cases with information at the 3 time points. For MFC analysis, PB samples were stained using a four-color direct immunofluorescence technique that allowed the quantification and characterization of T, NK and DC cells, including cell cycle analysis.The percentage of PB T cells in total PB cellularity was stable from baseline vs 3 vs 9 cycles of LenDex (22% vs 21% vs 21%; respectively, NS), with similar results also obtained for T CD4 (12% vs 11% vs 9%; respectively, NS) and T CD8 (8% vs 6% vs 8%; respectively, NS) cells. NK cells were slightly increased after 9 cycles of LenDex for both the CD56dim (4.1%, 3.4% and 6%; respectively; NS) and CD56bright (0.05%, 0.04% and 0.15%; respectively; NS) NK cell compartments. Similarly, the percentage of DC slightly increased along treatment, especially for plasmacytoid DC (0.2% at baseline vs 0.4% after 9 cycles; p=0.09). However, when a more detailed immunophenotypic characterization of T and NK cells was carried out significant differences emerged following LenDex treatment (Figure 1A). Accordingly, after 3 and 9 cycles of LenDex both T CD4 and CD8 cells showed increased expression of activation markers such as CD69 (p=.03), CD25 (p=.02 and NS, respectively), CD54 (p<.001), CD28 (p≤.03) and CD120b (p≤.01), together with increased production of IFNγ (p=.03) and IL-2 (p=.1 and p=.008, respectively). Interestingly, after induction therapy an up-regulation of chemokine receptors related to the Th1 (CCR5; p<.001) but also Th2 (CCR4; p≤.002) immune response was detectable in CD4 and CD8 T cells. T CD4 cells displayed a clear maturation into a central memory phenotype following LenDex treatment (38% at baseline vs 50% and 66% at 3 and 9 cycles, respectively; p<.001) while T CD8 cells displayed an increased effector memory phenotype (44% vs 59% vs 62%; p=.004). Further analysis showed increased expression of HLA-DR (p≤.008), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.03), and the adhesion molecules CD11a (p'.006) and CD11b (p≤.004) both on NK (CD56dim and CD56bright) and T cells. No consistent changes were observed in other NK cell receptors, such as CD94 and the immunoglobulin like receptors CD158a, CD161, NKB1 (3DL1) and NKAT2 (2DL3). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs 3 vs 9 cycles of LenDex in T CD4 (0.05% vs 0.15% vs 0.16%; p<.001), CD8 (0.05% vs 0.11% vs 0.23%; p<.001) and NK cells (0.09% vs 0.17% vs 0.20%; p=.001). Finally, an unsupervised cluster analysis of the overall immunophenotypic profile obtained after 9 cycles of LenDex (Figure 1B) was able to discriminate two groups of patients (A and B). Interestingly, within the group with higher activation profile (A) 50% of patients achieved ≥VGPR vs 23% in group B (p=.2).In summary, these preliminary results show that in high risk SMM patients the combination of lenalidomide and dexamethasone modulates PB T and NK cells, with increased activation status that may contribute to disease control. [Display omitted] Disclosures:Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria. Oriol:Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. Hernández:Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria, Speakers Bureau.

Comparison of indirect and direct immunofluorescence for the detection of Respiratory Syncytial Virus

The respiratory syncytial virus (RSV) is the major cause of respiratory infection in children, with bronchopneumonia and bronchiolitis. In the adult the clinical manifestation is lighter than in infant, however, in the case of immunocompromised patients (transplant recipients or patients in immunosuppressive treatment) infection can lead to death. Immunofluorescence is a diagnostic procedures which can detect RSV infection and the presence of viable virus in the biological sample of the patient. In this study, indirect and direct immunofluorescence techniques using monoclonal antibodies directed to RSV have been compared. For the comparison HEP2 shell vials, that are susceptible to RSV, were used.These cells were infected with different virus titres, ranging from 102 to 10-3 TCID50. Subsequently, different primary or Fluorescein isothiocyanate antibody dilutions were tested (1:40, 1:80, 1:160), for indirect and direct immunofluorescence evaluation, respectively, at three incubation periods after infection (48h, 72h, 96h). Indirect immunofluorescence showed a sensitivity of 101 TCID50, with a cells positive detection even at lower dilutions up to 10-1 TCID50 at 72 hours. For direct immunofluorescence, the same sensitivity was observed with 1:40 antibody dilution at 72h, with a detection limit of 10-1 TCID50. Indirect immunofluorescence showed optimal RSV detection at 72h after infection with 1:80 primary antibody dilution, with a sensitivity of 101 TCID50. An equivalent sensitivity has been observed with direct immunofluorescence at 72h, 1:40 antibody dilution, thus representing an advantage in terms of time.

Phagocytosis by normal polymorphonuclear leukocytes of immune complexes from serum of patients with Felty's syndrome and rheumatoid arthritis with special reference to IgE immune complexes.

Sera from patients with Felty's syndrome, rheumatoid arthritis (RA) and controls were investigated for the presence of immune complexes (IC) using phagocytosis by normal polymorphonuclear leukocytes and direct immunofluorescence technique. IC visible as large cytoplasmic inclusions were seen in 19 of 24 cases of Felty's syndrome, 3 of 16 cases of RA, and all 3 patients with extraarticular manifestations, and none of 21 control sera. IC containing IgG, IgA and complement C3 were found in nearly all positive cases. IgM IC were found in only 8 of the Felty's syndrome cases, IgE in 5 and beta-2-microglobulin in one case, respectively. A tendency to increasing number of large inclusion positive cells in vitro was found inversely correlated to the number of circulating leukocytes in the Felty patients at the time of serum sampling. In contrast, small cytoplasmic inclusions were found both in Felty's syndrome and RA patients and in some of the controls, and IgG and C3 were the most frequent constituents in these cases. As these inclusions were found in all groups it may have little significance. IgE IC as determined by a PEG precipitation technique were positive in the same 5 cases of Felty's syndrome with IgE containing inclusions, and in one case of RA with extraarticular manifestations. The complexed IgE amounted to about 3% of the total serum concentration of IgE. Phagocytosed IC may be involved in the pathogenesis of neutropenia and contribute to the inflammatory processes in Felty's syndrome.

Investigação de Campylobacter fetus e Tritrichomonas foetus na mucosa prepucial de touros da região do Médio Paraíba, RJ

Trinta e nove touros provenientes de propriedades de pecuária leiteira (n=9) e de pecuária de corte (n=30), situadas na região do Médio Paraíba, Rio de Janeiro (RJ), foram investigados para a presença de Campylobacter fetus e Tritrichomonas foetus. Para a pesquisa de Campylobacter, amostras de esmegma foram coletadas e submetidas à técnica de cultivo e isolamento e amostras de lavado prepucial ao teste de Imunofluorescência Direta (IFD). Para a pesquisa de Tritrichomonas, foi utilizada a técnica de exame direto a partir de lavado prepucial. Foi observada a presença de C. fetus em 14 amostras (35,9 %), por meio da IFD, e o isolamento de C. fetus, subespécie venerealis, foi obtido a partir de quatro amostras (10,3%). T. foetus não foi identificado nas amostras investigadas. A alta freqüência de C. fetus observada nos animais investigados sugere a presença da campilobacteriose na região do Médio Paraíba, em rebanhos com problemas reprodutivos.

Induction of Upregulation and Downregulation of the T-Cell Activation Marker CD98 in Patients Undergoing Contrast-Enhanced CT with Iodinated Non-Ionic Dimeric Contrast Medium

ObjectiveThis study was designed to determine prospectively the expression of the multifunctional CD98 protein in peripheral white blood cells in patients receiving iodinated contrast media (CM) for a computed tomography (CT) examination.Materials and MethodsIn 12 adult patients that received non-ionic dimeric CM (iosimenol or iodixanol), the expression of CD98 was analyzed from samples of peripheral white blood cells obtained prior to, one hour, and 24 hours after CM injection by the use of flow cytometry analysis and the use of the direct immunofluorescence technique.ResultsOverall, expression of CD98 was significantly downregulated 24 hours after CM injection (51.9%±10.8% vs. 38.8%±16.9%; p < 0.04). Patients that received iosimenol exhibited a more pronounced but not significant decrease of CD98 expression both one hour and 24 hours after CM injection. In an analysis of specific patient responses, CD98 downregulation occurred in eight patients. In two patients, CD98 was upregulated, and in the remaining two patients, expression remained unchanged. No patient acquired an adverse CM reaction.ConclusionThis is the first demonstration that CM may be a regulator of CD98 expression. To determine if upregulation is associated with an increased risk for the acquisition of an adverse CM-induced hypersensitivity reaction and if downregulation is associated without a risk for the acquisition of an adverse CM-induced hypersensitivity reaction, further studies with a larger population of patients are required.

Neumocistosis en pacientes venezolanos: diagnóstico y epidemiología (2001-2006)

The objective of this work was to investigate the epidemiology of pneumocystosis in Venezuelan patients utilizing a retrospective study during a six year period. One hundred and twenty nine clinical samples collected from patients with AIDS, cancer and non-AIDS-non-cancer low respiratory tract infection patients were processed by direct immunofluorescence technique. Pneumocystosis was diagnosed in 30 patients with a general frequency of 23.3%, which varied according to the patient's group: 36.6% in AIDS patients, 38% in cancer patients, and 10.4% in non-AIDS-non-cancer low respiratory tract infection patients. This study demonstrated the existence of differences in pneumocystosis frequency related to the patient's underlying disease, and that the illness is an important health problem in immunocompromised patients in Venezuela. Pneumocystosis must be suspected in non-immunocompromised patients with signs and symptoms of low respiratory tract infection, and the study of this illness must include COPD and cancer patients. Direct immunofluorescence is a useful technique for pneumocystosis diagnosis, however, it requires an optimal sample and skilled personnel in the laboratory.

Oral manifestations of systemic and cutaneous lupus erythematosus in a Venezuelan population

The aim of this study was to characterize oral lesions in patients with systemic and cutaneous lupus erythematosus (LE) in a Venezuelan group. Ninety patients with LE were studied. Oral biopsies were taken from patients who showed oral mucosal involvement. Tissue samples were investigated with histology and direct immunofluorescence techniques for the presence of immunoglobulins G, M, A and complement factor C3. In 90 patients with LE, 10 patients showed oral lesions related to the disease. Sixteen lesions were investigated. Oral ulcerations accompanied by white irradiating striae occurred in five patients, erythema was observed in five patients and a white homogeneous plaque in one patient. Fifteen lesions demonstrated vacuolar basal degeneration and 12 thickening of the basement membrane histologically. Direct immunofluorescence was negative in three samples. These findings corroborated that ulcers are not the only manifestation of LE in the oral mucosa. Clinical and histological examinations are significant as immunoproteins are not always found on the oral sample.

Role of direct immunofluorescence on Tzanck smears in pemphigus vulgaris

The Tzanck smear is a simple, sensitive, and rapid test to diagnose pemphigus vulgaris (PV), a life threatening autoimmune blistering disorder. The presence of acantholytic cells in cytology is indicative of but not specific for PV. Hence, a direct Immunofluorescence (DIF) test to demonstrate immunoglobulin deposits on the acantholytic cells would make the Tzanck test more specific, in addition to being a rapid test. Twenty untreated patients with PV confirmed histopathologically were enrolled to evaluate the efficacy of using DIF technique using IgG on Tzanck smear samples. The DIF smears were compared with DIF on skin biopsies in the same patient. This prospective pilot study approved by the institutional ethics committee was carried out in a tertiary health care hospital in a developing country. Of the 15 patients presenting within 3 mo of onset of the illness, 40% (n = 6) showed DIF positivity on Tzanck smear, when compared with 46.67% (n = 7) on skin biopsy. On the other hand, of the five patients presenting beyond 3 mo of their illness, only 20% (n = 2) showed positivity on Tzanck, when compared with all 100% (n = 5) on skin biopsy. The study, thus, suggests that DIF on skin biopsy is comparable to biopsy in diagnosing early PV. This preliminary study proposes that the use of DIF on Tzanck smear is a simple, rapid, painless, and user-friendly out-patient procedure for the diagnosis of early PV, even for relatively inaccessible lesions in the oral cavity and flexural regions. This methodology would be of great help in outlying and rural facilities lacking proper histological equipment, thus avoiding the need for a surgical or punch biopsy or heavy investment in laboratory equipment and expertise. Probable reasons for DIF negativity on Tzanck smears are also discussed.

Detection of pemphigus autoantibodies in healthy relatives of Turkish patients with pemphigus

Pemphigus autoantibodies have been reported in healthy relatives of pemphigus patients suggesting a genetic predisposition in the pathogenesis of the disease. To test for the presence of pemphigus autoantibodies in healthy relatives of Turkish patients of pemphigus. The study group comprised 45 pemphigus patients, 75 unaffected family members and 47 healthy individuals in the control group. Direct and indirect immunofluorescence techniques were performed to determine the presence of pemphigus autoantibodies. By indirect immunofluorescence staining, circulating pemphigus autoantibodies were found in 26.7% of the relatives and in only two of the controls (P value = 0.0001). A direct immunofluorescence technique revealed positive results in three (4%) of the relatives and none of the controls. The presence of pemphigus autoantibodies in clinically healthy relatives indicates that genetic predisposition is necessary but not sufficient for the development of clinical disease.

Role of Electron Microscopy in the Diagnosis of Ocular Mucous Membrane Pemphigoid

To evaluate the role of electron microscopy (EM) for the diagnosis of ocular mucous membrane pemphigoid (MMP) among patients with cicatrizing conjunctivitis. Retrospective case series. One hundred twenty-eight patients with cicatrizing conjunctivitis referred for the evaluation of possible ocular MMP between January 1985 and February 2002 who underwent conjunctival biopsy and evaluation with EM and direct immunofluorescent (DIF) techniques. The diagnosis of each patient was based on DIF techniques. The reproducibility of EM readings was measured by assessing intraobserver and interobserver variability. The diagnosis from EM was compared to the DIF diagnosis from the same patient to evaluate the validity of the EM diagnosis. Sensitivity, specificity, positive predictive value, and negative predictive value of EM for diagnosing ocular MMP. One hundred twenty-six of 128 conjunctival biopsies were available for evaluation of EM findings. The percent agreement between 2 readings from the same observer was 92%, and the percent agreement between 2 independent observers was 78%. The sensitivity and specificity of EM for the diagnosis of ocular MMP were 51% and 72%, respectively. The positive predictive value of EM for the diagnosis of ocular MMP was 49%. The reproducibility of EM findings was good as indicated by high percent agreement for both intraobserver and interobserver measurements. However, the sensitivity, specificity, and positive predictive value of EM for the diagnosis of ocular MMP was low. It seems that EM has limited usefulness in the diagnosis of ocular MMP.

Epidemiology and Clinical Characteristics of Respiratory Syncytial Virus Infections in Jordan

This study was carried out to determine the prevalence, seasonal distribution of RSV, the signs and symptoms associated with it in Jordan. A total of 200 nasopharyngeal aspirates were obtained from hospitalized children (below 2 years old). RSV was detected in 12.5% of patients using direct immunofluorescence technique. Most infections were associated with bronchilolitis, and higher rates of hypoxemia, retractions, tachypnea, hyperinflation and interstitial infiltrates in 1 to 3 months old children. RSV showed a clear temporal periodicity. The epidemic began in December and disappeared in March with a peak of incidence during February 2003 and January 2004. The seasonal distribution showed a significant correlation with temperature, rainfall and relative humidity. This study provides further information on RSV epidemiology which could help in planning of prevention and control programs in Jordan, distinguishing RSV infections on the basis of the clinical picture and considering RSV between December and March each year.

Aislamiento e identificación de pestivirus obtenidos de alpacas (Lama pacos) y llamas (Lama glama) de la Región Metropolitana, Chile

SUMMARY The natural habitat for more than 90% of the domestic South American camelids (SAC) in Chile, alpaca (Lama pacos) and llama (Lama glama), is located between 11° and 21° South latitude at 3,800 and 5,000 ms of altitude. Lately, alpacas and llamas have been introduced to other geographic parts of the country where they are in contact with domestic ruminants, making likely infection with BVDV, present in cattle, goats and sheep. The BVDV includes two species, BVDV genotype I (BVDV I) and BVDV genotype II (BVDV II), which along with the border disease virus (BDV) and classical swine fever virus (CSFV) conforms the Pestivirus genus of the Flaviviridae family. This study evaluates the hypothesis that SAC introduced to the Metropolitan Region (MR) of Chile are infected with pestiviruses. In order to perform viral isolation, samples were taken from 80 SAC (42 live alpacas, 35 live llamas, 2 dead llamas and 1 aborted foetus of llama), coming from 4 flocks suspected to be infected with pestivirus. The samples were inoculated in primary culture of bovine foetus lung cells (free of BVDV), passing each sample 5 times, and were then analyzed by direct immunofluorescence and indirect immunoperoxidase techniques to detect the presence of pestivirus antigens. For molecular characterization, a fragment of the 5’-untranslated region (5’-UTR) of RNA of the isolates was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and treated with restriction enzymes Pst I, Bgl I and Xho I in order to identify species of viruses. The results show that 18 SAC (10 alpacas and 8 llamas from the 4 studied flocks), were infected with pestivirus. All isolates were non cytopathogenic. BVDV I was isolated from 6 alpacas while BVDV II was isolated from 4 alpacas and 8 llamas . The viral samples were obtained from 8 healthy alpacas, 2 alpacas with abortion, 5 healthy llamas, 2 llamas with abortion and 1 dead llama without clinical history. It is concluded that alpacas and llamas from the MR of Chile are infected with BVDV I and BVDV II.

Identificación del antígeno del virus de la fiebre porcina clásica en excretas porcinas sólidas y líquidas en una granja del estado de Guanajuato, México

The purpose of this study was the detection of Classical Swine Fever virus (CSFv) in organic waste matter. A total of 40 samples from collection basin sludge, solid faeces and separated liquid phases from eight farms with faeces treatment systems based on a liquid and solids separation process in Central Mexico, were evaluated. All the samples were tested by direct immunofluorescence, direct immunoperoxidase and capture ELISA techniques to determine the presence of CSFv. The results from all samples were negative to immunoperoxidase and mmunofluorescence tests. Two of the samples showed positive results to a capture ELISA test (5.0 %). Both samples were collected in the same farm in the State of Guanajuato, one from the sedimentation basin and the other from separated solid matter. The CSFv may be isolated from solid swine excrement and from solid waste matter in the basin as well. The capture ELISA test proved to be more sensitive, due to its ability to detect specific CSFv proteins. Immunoperoxidase and immunofluorescence tests failed to detect the virus.

ZAP-70 Expression in T Cells of B-Cell Chronic Lymphocytic Leukaemia: Correlation with Negative Prognostic Factors of the Disease.

Several studies demonstrated that ZAP-70, a tyrosine kinase which plays a critical role in T cell antigen receptor signaling, is expressed in cells of B cell chronic lymphocytic leukemia (B-CLL) in correlation with unmutated status of immunoglobulin genes, progression of the disease and overall survival. Recently, it has been reported a correlation among ZAP-70 expression in T cells of CLL and CLL Zap-70 levels, clinical stage and disease progression. To analyse the functional capacity of the T residual component in the B-CLL in this study we evaluated ZAP-70 expression in peripheral T cells from patients with B-CLL and from healthy volunteers using a direct immunofluorescence technique by flow cytometry (FACSCAN, Becton Dickinson). Analysis was performed using CellQuest software and results were expressed using mean fluorescence index (MFI) calculated as ratio between mean channel of sample and mean channel of negative control.Sixty two B-CLL patients and ten normal subjects entered the study, 35/62 patients were males and 27 females. Our results showed a higher ZAP-70 expression in T cells from B-CLL compared to normal T cells, with a median MFI value of 14 (range 4,2–65) and 11 (range 9,2–14,6) respectively (p=0,045). Moreover, using a cut-off of 11 (that represents the median MFI value in normal T cells) we defined two groups of B-CLL cases: patients with high expression (MFI > 11) and patients with low expression (MFI ≤ 11). No differences among the various stages were recorded in the two groups, in fact 25/50 (50%) ZAP-70high T-cell case were stage A, 8/50 (16%) were stage B and 17/50 (34%) were stage C, and 9/12 (75%) ZAP-70low T-cell cases were stage A, 1/12 (8%) were stage B and 2/12 (16%) were stage C. High expression of ZAP-70 in T cells seems to correlate with negative prognostic factors, in fact our results showed a higher incidence of CD38 and of unmutated immunoglobulin genes in ZAP-70high T-cell group with respect to ZAP-70low T-cell cases: 24/45 (53%) vs 3/11 (27%) and 22/29 (76%) vs 3/7 (42%) however these associations were not of statistical significance, probably due to the small numbers of patients analysed. On the contrary a significant correlation was recorded with level of expression in T cells and presence of ZAP-70 in B cells, indeed ZAP-70 was expressed in B lymphocytes of 37/50 (79%) patients from ZAP-70high T-cell group, while only in 5/12 (41%) cases among ZAP-70low T-cell group (p=0,04).The meaning of ZAP-70 over expression in T cells of B-CLL is unknown. Some studies suggest that it may be indicative of a state of cell activation, however very preliminary data on 4 patients show a notably lower constitutive phosphorilation state compared to the one observed in T cells of healthy volunteers. Besides, our data don't show a significant increase of the global phosphorilation pattern after T cell stimulation with PHA, even if the CD3 e resulted strongly phosphorilated, suggesting a reduced responsiveness of T cells to mitogenic stimuli.In conclusion, ZAP-70 over-expression in T cells from B-CLL seems to identify a subgroup of patients with a more aggressive disease, and moreover we hypothesize that it may be the expression of alteration of T cells functionality associated to the disease.

Isolation of pathogenic Legionella species and legionella-laden amoebae in dental unit waterlines

Legionella released into the air during treatment are a potential source of infection. Water stagnation in dental unit waterlines (DUWLs) creates biofilms and promotes the proliferation of these micro-organisms. This study investigated the presence of amoeba infected with legionella, L. pneumophila and other pathogenic Legionella species in a dental teaching hospital. Water samples were collected in the morning and afternoon from 99 dental units and 16 taps connected to the municipal water supply. Samples were plated on selective media and tested for legionella using the direct immunofluorescent antibody technique and the latex agglutination test. Legionella were found in 33% of the DUWLs and in 47% of the mains taps supplying these units. Legionella-laden amoebae occurred in one mains tap sample and in 20% of DUWLs in a clinic of the teaching hospital. L. micdadei was the predominant species isolated from this clinic. L. pneumophila serogroups 2-14 predominated in the mains water, whereas L. pneumophila serogroup 1 was found in approximately half of the contaminated DUWLs and mains taps irrespective of the time of sampling. Pathogenic Legionella species seeded by municipal water into DUWLs is a potential source of legionella infection for both dental personnel and patients during prolonged dental treatment. This problem is compounded by the presence of legionella-laden amoebae which may contain levels of organism well within the infective dose. The interaction of legionella with amoebae is an important ecological factor that may significantly increase the risk of legionellosis, and thus should be given further consideration in the refinement of risk assessment models.

Cellular ultrastructural changes of bone marrow of patients with hemorrhagic fever with renal syndrome

To observe cytopathogenic effect of Hantaan virus (HV) on cultured human bone marrow cells. Light and transmission electron microscopy and direct immunofluorescent technique were applied to study cellular structure especially ultrastructural changes of bone marrow cells from patients with Hantaan virus infection. Bone marrow cells of one healthy volunteer were also studied as control. The antigen of HV was found in bone marrow cells of 20 of 27 HFRS patients by the aid of direct immunofluorescent technique. It was found that the granulocytes had the highest percentage of HV antigen positive cells (76%), followed by monocytes (65%), lymphocytes (40%), megakaryocytes (20%) and the lowest was found in erythrocytes (3.7%). The injury of cell membrane after infection with HV was significantly more severe than that in the control group under the light and electron microscopy. This study demonstrated that HV could attack human bone marrow cells and cause cytopathogenic effect on them.

Increased peripheral blood B-cells expressing the CD5 molecules in association to autoantibodies in patients with lupus erythematosus and evidence to selectively down-modulate them

The present investigation has been undertaken to analyze absolute and relative CD5+ B-cell numbers in patients with lupus erythematosus (LE), and concomitant B-CLL, and to monitor them under therapy. Peripheral blood lymphocytes of LE-patients, and healthy controls were analyzed by flow cytometry and direct immunofluorescence technique. Patients were treated with low-dose methotrexate (MTX). Before and during MTX treatment laboratory monitoring has been done. LE-patients had increased percentages of CD5+CD19+ as compared to controls ( p < 0.0002), the absolute number of CD5+ B-cells was equal in controls and patients. Autoantibodies were positively correlated to the number of CD5+ B-cells in LE-patients. In a total of 140 LE-patients one male patient suffered from both LE and B-CLL (0.7%). He had increased absolute and relative CD5+ B-cells. MTX induced significant decrease of both total B-cell numbers, and CD5+ B-cells. The decrease of CD5+CD19+ cells was more pronounced than the decrease of total B-cells. Apoptosis rate increased in parallel to the drop-down of elevated CD5+CD19+ cells. Peripheral T-cell subsets remained stable under low-dose MTX. Both absolute and relative numbers of CD5+CD19+ cells should be taken into account in patients with LE. MTX seems to decrease B-cells, and preferentially to down-regulate B-cells expressing the CD5 molecule, which opens new therapeutic options and cell biological activities. The mechanism is unclear but apoptosis induction seems to be likely.