Fitoplancton en lagunas de estabilización

Describe el rol de las algas en los sistemas de lagunas de estabilización de líquidos cloacales y en el reuso de aguas residuales para la acuicultura, formando parte de la cadena alimentaria y proveyendo oxígeno.En el estudio del fitoplancton se analizaron las lagunas de estabilización de Saladas y Mercedes, de la provincia de Corrientes.Incluye gráficos y bibliografía

Fitoplancton en lagunas de estabilización

Describe el rol de las algas en los sistemas de lagunas de estabilización de líquidos cloacales y en el reuso de aguas residuales para la acuicultura, formando parte de la cadena alimentaria y proveyendo oxígeno.En el estudio del fitoplancton se analizaron las lagunas de estabilización de Saladas y Mercedes, de la provincia de Corrientes.Incluye gráficos y biliografía

Bestatin-mediated inhibition of leucine aminopeptidase may hinder HIV infection.

Bestatin, an inhibitor of leucine aminopeptidase (LAPase), significantly decreased HIV infection as reflected by a reduced number of positive immunofluorescent cells, p24 levels, reverse transcriptase activity and the number of proviral copies found in Bestatin-treated cells. Cellular and extracellular LAPase activity in infected cells was higher than the LAPase activity found in uninfected cells. However, cellular and extracellular LAPase activity as well as total protein kinase C activity was lower in Bestatin-treated cells. Conversely, the incubation of human lymphocytic HUT78 cells with LAPase promotes HIV infectivity. The possible role of LAPase in the pathophysiology of HIV was assessed by determining LAPase serum levels in HIV infected patients. LAPase activity levels were three orders of magnitude greater in sera obtained from HIV patients than those detected in sera of uninfected individuals. Although Bestatin reduced HIV infection, a moderate decrease in the reverse transcriptase activity of chronically-infected H9 human T-lymphocytic cells was observed. Based on the higher levels of LAPase present in the serum of HIV patients and on the combined inhibitory effect of Bestatin on LAPase and on protein kinase C activities, we suggest that LAPase may play an important role in the early events of HIV infection such as viral entry.

Cell surface marker evaluation of infant Macaca monkey leukocytes in peripheral whole blood using simultaneous dual-color immunophenotypic analysis.

Cross-reactivity between several commercially available mouse antihuman monoclonal antibodies (mAbs), conjugated to phycoerythrin (PE) and fluorescein isothiocyanate (FITC) fluorochromes, and peripheral blood leukocyte surface antigens, has been established in infant cynomolgus (Macaca fascicularis) monkeys using whole blood lysis, and two-color, PE and FITC flow cytometric analysis. With the exception of the CD8 marker, the bivariate dot-plot patterns for all other markers were similar in infant monkeys and in humans. For the CD8 marker, however, a CD8+CD2- population of cells was observed in the majority of monkeys tested (10 out of 12). The number of CD8+CD2- cells was higher (13%) in infant monkeys compared to the 3% reported for adult human blood. The mean percentage and absolute numbers for the cell surface markers identified with the human mAbs CD2 (FITC, Ortho, Paritan, NJ), CD4 (PE, B-D, Mountain View, CA), and CD8 (PE, B-D) when these were combined with a series of PE- or FITC-labelled human mAbs were similar across all combinations tested. Statistically significant differences were observed between male and female monkeys for the mean percentage levels of CD4 (females > males) and for the CD4/CD8 ratio (females > males). Such gender differences need to be taken into consideration when infant cynomolgus monkeys are used as models for chemical-induced immunotoxicity studies. Measurement of the interleukin-2 (IL-2) and transferrin proved to be useful in monitoring in vitro cellular activation in infant cynomolgus and possibly in rhesus (M. mulatta) monkeys.

Blood monocytes from most human immunodeficiency virus type 1-infected patients do not carry proviral DNA.

In blood, the CD4+ T cells of patients with human immunodeficiency virus type 1 (HIV-1) harbor HIV-1; however, whether the CD4+ blood monocytes carry the virus is controversial. Tissue macrophages are known to be infected. To determine in blood monocytes from HIV-1-seropositive patients contain HIV-1, we separated monocytes and T-cell subsets by using monoclonal antibodies bound to magnetic beads and by monocyte adherence to glass. Monocytes were cultured with macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3. After 14 days in culture, cells were analyzed for the presence of HIV-1 antigen and multinucleated giant cells (MGCs). Freshly isolated cell subsets were analyzed for HIV-1 proviral DNA by PCR with modified env (SK68i and SK69i2) and gag (SK145i and SK150) primers. We found that (i) monocytes cultured without depletion of CD4+ T cells (11 of 11 patients) were HIV-1 antigen positive and showed dramatically increased spontaneous formation of MGCs (ii) monocytes cultured after depletion of CD4+ T cells (three experiments) were HIV-1 antigen negative and showed markedly decreased MGC formation, and (iii) in specimens from 14 patients subsequently analyzed by PCR, purified CD4+ T cells were positive for HIV-1 proviral DNA in all patients. In 11 of 14 patients (79%), the monocyte fractions were HIV-1 proviral DNA negative, while in the remaining 3 patients, the monocytes were positive for HIV-1 proviral DNA. In conclusion, the major reservoir for HIV-1 infection in human peripheral blood is the CD4+ T cell (14 of 14 cases).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of HIV-1 replication by daunorubicin.

Members of the anthracycline family of drugs such as daunorubicin (DNR) are currently being employed in cancer therapy, treatment of Kaposi's sarcoma and lymphomas in HIV-1 infected patients. These drugs may have an anti-viral activity against HIV-1. Experiments conducted on U937 (a promonocytic cell line), peripheral blood monocytes (without T cells) and infected with HIV-1 showed anti-viral activity at nanogram/ml concentrations, without affecting cell proliferation and cell viability. Treatment of Hut 78 cells (a T cell line) with DNR was not as effective in inhibiting HIV-1 replication when compared to the anti-HIV activity in cells of the monocyte lineage.

Suppression by HIV of IL-1 and IL-6 secretion in accessory cells: AC function defect partially corrected with exogenous IL-1 and IL-6.

To determine the effect of HIV infection on the accessory cell function of monocytes we measured the ability of HIV-infected monocytes to restore PHA-induced and soluble anti-CD3-induced T cell blastogenesis. These T cells were highly purified and depleted of monocytes (< 0.5%) and activated T cells. Monocytes were isolated using gelatin-fibronectin-coated flasks (< 1% T cells) and after 4 days in culture with granulocyte/macrophage-colony stimulating factor, they were infected with HIV. Accessory cell (AC) function was tested 2 and 7 days later, employing autologous cryopreserved T lymphocytes. Monocytes infected with HIV for 2 days lacked the ability to permit phytohemagglutinin (PHA) and anti-CD3-induced T cell blastogenesis. Noninfected monocytes restored the proliferative response of purified T cells. Interleukin 1 (IL-1) and interleukin 6 (IL-6) levels in culture supernatants were low when compared to cultures with noninfected AC. Preincubation of monocytes with human anti-HIV neutralizing antibodies did not restore either of the responses. AC treated with heat-inactivated HIV had normal accessory cell function. The addition of IL-1 and/or IL-6 partially restored the AC function for PHA stimulation, but not for anti-CD3 stimulation. We conclude that HIV infection of monocytes suppressed their accessory cell function in the T cell blastogenesis assay. The response was partially restored with IL-1 and/or IL-6, suggesting that HIV infection down-regulated the monocyte production of both cytokines.

A simultaneous three-color T cell subsets analysis with single laser flow cytometers using T cell gating protocol. Comparison with conventional two-color immunophenotyping method.

We describe a method for simultaneous analysis of CD3, CD4, and CD8 positive cells from whole blood utilizing single laser flow cytometers. All three T cell values are attained from a single test tube. CD4 and CD8 positive cells are identified only if they are CD3 positive. Thus the values obtained by this method for T helper/inducer and T cytotoxic/suppressor cells can be reported directly as a percentage of T lymphocytes. Analysis for CD4 and CD8 positive cells is accomplished, by first gating on CD3 positive T lymphocytes, hence the approach is referred to as a T gating method. As the third dye, conjugated to anti-CD3 monoclonal antibodies (MAbs), we utilized peridinin chlorophyll protein (PerCP), a new red fluorochrome. The proposed method may prove to be practical for monitoring disease progression in AIDS, where longitudinal T helper/inducer and T cytotoxic/suppressor cell enumeration must be performed unambiguously by a simple, reproducible, and fast method.

Reticulo-fibroblastoid stromal cell progenitors (CFU-RF) in murine bone marrow.

The hemopoietic inductive microenvironment (HIM) of the bone marrow is responsible for secretion of growth factors that regulate hemopoiesis. It is composed of an extracellular matrix and a complex variety of cell types with a range of functions related to blood cell development. In order to understand how such a complex system operates, it will first be necessary to determine the role(s) of the integral parts. Several of the stromal cell types have been identified morphologically in various culture systems, and some of their functions have been elucidated. We have identified a new stromal cell type in mouse bone marrow that appears similar if not identical to its human counterpart. When bone marrow cells are placed in methylcellulose/plasma clot culture with phytohemagglutinin-stimulated human leukocyte-conditioned medium in the presence of bovine calf serum (BCS), mercaptoethanol, and hydrocortisone, extensive branching colonies develop within 14 days. These "reticulo-fibroblastoid" (RF) colonies arise from a putative reticulo-fibroblastoid colony-forming unit (CFU-RF) stem cell, and many become adipocytic by day 14; the addition of fresh medium, methylcellulose, and BCS on day 7 inhibits this change. The batch of human citrated plasma used in the culture system and the type and source of stimulating factor all influence the number of RF colonies that develop as well as the percent of colonies that become adipocytic. Whether this adipogenesis represents functional maturity or terminal differentiation is not yet known. Information gained on the role of these RF cells in normal and impaired hemopoiesis should contribute to the elucidation of the complicated interactive role of the microenvironment in the support and modulation of hemopoiesis.

Erythroid lineage-specific activity in conditioned medium derived from cloned human marrow stromal cells (CFU-RF).

Using long-term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self-renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU-RF. In this paper, a method is described for subculturing cells from individual CFU-RF-derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage-specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose-response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony-stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.

Detection of surface and cytoplasmic CD4 on blood monocytes from normal and HIV-1 infected individuals.

CD4 on blood monocytes is generally regarded as being found on a subset of blood monocytes. However, our results show that all monocytes are CD4+ but the number of molecules per cell is lower than T cells. We have performed immunofluorescent (flow cytometry, microscopy) analysis of monocytes from normal donors and HIV-1-infected patients using anti-CD4 (Leu-3a) and the monocyte-specific marker anti-CD14 (Leu-M3) monoclonal antibodies. Specifically: (1) 91% of monocytes from normal individuals show dual positivity for CD14 and CD4 (n = 14) as measured by flow cytometry; (2) 76% of monocytes expressed surface bound CD4 and CD14 when an enhanced two colour immunofluorescence microscopic technique was employed; (3) all CD14+ monocytes stained with an intensity of 3+(-)4+ for cytoplasmic CD4. Few monocytes were CD14- and CD4+. Cell surface CD4 expression was blocked with unconjugated anti-CD4 prior to staining; (4) the staining intensity for cytoplasmic CD4 in T cells was negligible; (5) CD4 expression on monocytes obtained from patients was as observed with normal individuals. The conclusion drawn is that all monocytes are CD4+ and the CD4 expression in monocytes is mainly cytoplasmic. Thus, all monocytes are potentially infectable with HIV-1.

Absence of a bleeding tendency in severe acquired von Willebrand's disease. The role of platelet von Willebrand factor in maintaining normal hemostasis.

A 67-year-old male with a prolonged activated partial thromboplastin time (APTT) of 43 seconds (normal, 25-40 seconds) was found to have laboratory features of von Willebrand's disease and IgA myeloma but had a normal bleeding time and no bleeding tendency. Plasma Factor VIII coagulant activity (F.VIII:C) was 80 U/L (0.08 U/mL), Factor VIII antigen (F.VIII:Ag) 70 U/L (0.07 U/mL), and von Willebrand's factor antigen (vWF:Ag) 50 U/L (0.05 U/mL) and ristocetin cofactor (vWF:RiCoF) 10 U/L (0.10 U/mL). The platelet vWF:Ag level was normal, whereas both platelet lysate and plasma vWF high molecular weight multimers were decreased. Patient plasma had no inhibitory effect on either F.VIII:C or vWF:RiCoF. However, when patient plasma was incubated with normal plasma, crossed immunoelectrophoresis for vWF:Ag demonstrated the presence of immune complexes. Infusion of 1-desamino-8-D-arginine vasopressin led to a transient correction of the plasma vWF:Ag multimer pattern. The survival of all components of vWF/F.VIII was decreased, as also occurred after cryoprecipitate. The levels of plasma F.VIII/vWF increased as the IgA values decreased after chemotherapy, whereas the platelet high molecular weight multimers remained decreased. The data suggest that the plasma vWF/F.VIII deficiency results from complexing of the IgA myeloma protein with vWF, resulting in premature clearance of the vWF/F.VIII complex. The absence of clinical bleeding likely results from the combination of a normal platelet vWF:Ag level and persistence of intermediate molecular weight vWF multimers.

Phytohemagglutinin mitogenic response of normal individuals vaccinated with hepatitis B vaccine.

The phytohemagglutinin (PHA) blastogenic response of normal healthy individuals was studied before and after vaccination with hepatitis B surface antigen. The PHA response was suppressed 2 d after the first dose of vaccine but was not affected by the second and the third doses of vaccine. The suppressed PHA blastogenic response on day 7 was not enhanced by the addition of interleukin-2 or indomethacin even though an increase in cell number expressing CD25 was observed. The removal of CD4+ or CD8+ cells enhanced the PHA response but only on days 2 or 4 and not at other sampling times, which suggests that the suppression is mediated by CD4+ or CD8+ cells. The addition of interleukin-2 alone or with PHA did not reverse the suppression at any time tested. In vitro induction of suppressor cells was performed and was blocked by the addition of indomethacin at the time of culture initiation.

The role of CD4+ lymphocytes in the activation of non-specific suppressor cells by antigen.

Tetanus toxoid (TT) and hepatitis B surface antigen (HBsAg) can suppress lectin-induced responses. The suppression induced by TT is dose-dependent and can also down-regulate the induction of a blastogenic response by anti-CD3 and anti-CD4 monoclonals. In addition, TT can dampen the blastogenic response induced by phytohaemagglutinin (PHA) and anti-CD3. The cellular mechanism involved in the turning off of the blastogenic response was investigated by transferring cells treated for 5 days with TT to freshly obtained syngeneic cells and stimulation with PHA. The response of cultures that had received TT-treated cells was significantly lower than of those that had received cells treated with medium only. The removal of CD4+ cells at the induction phase of the suppression reversed the suppression, whereas the elimination of most CD8+ cells had no effect. We propose that CD4+ cells together with monocytes can dampen the specific and non-specific blastogenic responses.

Acute promyelocytic leukemia associated with a paraprotein that reacts with leukemic cells.

A 29-year-old woman developed acute promyelocytic leukemia during pregnancy. At diagnosis, immediately postpartum, she was found to have IgG kappa immunoglobulin on the surface of the leukemic cells as well as a monoclonal protein of IgG kappa specificity in her serum. These resolved with chemotherapy which induced a complete remission. Immunoglobulin gene rearrangement was not found in the leukemic cells, thus indicating that the blasts were not secreting the monoclonal protein. The authors believe that the patient had an autoantibody directed at myeloid cells which was amplified by the development of the leukemic process.

Residual amounts of glycoprotein Ib concomitant with near-absence of glycoprotein IX in platelets of Bernard-Soulier patients.

A study of the Bernard-Soulier syndrome in two unrelated families using different polyclonal antibodies in a sensitive immunoblot assay showed residual amounts of platelet membrane glycoprotein (GP) lb in the eight homozygotes, as well as the near-absence of GPlb beta and GPIX. The eight heterozygotes studied showed a double band pattern for GPlb and about half the normal level of GPlb beta and GPIX. Therefore, we conclude that the Bernard-Soulier syndrome is heterogeneous and is probably not due to gene deletions.

Origin of T lymphocyte colony-forming cells in cell populations depleted of sheep erythrocyte rosette forming cells.

Cell populations depleted of sheep erythrocytes (E) rosette-forming T cells (E-cells) contain cells capable of giving rise to T cell colonies. We have characterized the T cell colony-forming cell from human bone marrow, blood and tonsil E- cells using a T-cell colony assay. Depletion of CD2+, CD3+ or CD4+ cells from E- cells reduced colony formation by 70-100%. Removal of CD8+ cells did not reduce, but rather enhanced colony formation by 50% or more. The most effective reduction (100%) in colony formation was obtained with anti-CD4, indicating that CD4 is a marker of all colony-forming T cells. The CD4+ lymphocytes generated two types of colonies, types I and II, in the presence or absence, respectively, of CD8+ lymphocytes. Type I were small and compact, reached a peak on days 5-7, and contained CD4+ and CD8+ cells. Type II were large and diffuse, reached a peak on days 9-10 and contained CD4+ cells. In continuous culture of single type II colony cells, we observed a consistent increase of CD8+ cells. In one colony the combined percentage of CD4+ and CD8+ cells exceeded 100% (averaging 83% CD4+ and 72% CD8+), indicating the presence of dual markers on some cells. We suggest that colony forming T cells are CD2+ CD3+ CD4+, the CD4+ antigen being the most consistent marker of such precursor cells.

Quantitation of cell membrane glycoproteins in pathological conditions using a lectin-bound enzyme-linked immunosorbent assay (ELISA). Application to human platelets in the Bernard-Soulier syndrome.

Several techniques are available to study cell membrane glycoproteins in pathological conditions but these either lack sensitivity or require radiolabelled material or expensive apparatus. We have, therefore, developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) to study patients with the Bernard-Soulier syndrome (BSS), a hereditary platelet disorder characterized primarily, at the molecular level, by glycoprotein Ib (GpIb) deficiency. We have used the lectin wheat germ agglutinin to capture GpIb in the microtiter wells. Following incubation with monoclonal antibody AN51 which recognizes an epitope on GpIb, the immune complex was detected using the streptavidin-peroxidase-biotin complex. Platelet samples from 24 normal controls gave a mean value of 106% whereas in the six BSS patients the mean value was 14% and in the eight obligatory heterozygotes it was 78%. The technique is simple, inexpensive and sensitive and does not require the use of radioactive material. The assay method could be applied to quantitate other cellular glycoproteins where specific lectins and monoclonal antibodies are available.