First Report of Vinca Blight Caused by Phytophthora nicotianae in Northwestern Mexico.

Vinca (Catharantus roseus (L.) G. Don) is a common ornamental landscape plant. From July to September 2012, blighted and wilted vinca plants were found in retail stores, commercial nurseries, and urban landscape areas of Culiacan, Sinaloa, in northwestern Mexico. In several commercial nurseries and a retail store, incidence of the unknown disease on vinca plants ranged from 20 to 50%, resulting in significant economic losses. Symptoms of the disease started with a foliar blight, and if warm and wet conditions were present, the disease progressed, causing plant wilting and death. Surface-sterilized (0.5% NaOCl 1 min) diseased plant tissue was plated on V8 agar medium, and after 72 h of incubation at 25°C, white colonies of coenocytic mycelium were developed from the plated tissues. Isolates produced cottony colonies on V8 agar medium, grew well between 7 and 30°C (optimum of 25°C), and produced spherical, intercalary, and terminal chlamydospores (17 to 30 µm) and non caducous, papillate, spherical to ovoid sporangia of 30 to 39 × 21 to 31 µm. Based on these morphological characteristics, Phytophthora isolates were identified as Phytophthora nicotianae Breda de Haan (1,3). The identity of two representative isolates OV4 and OV11 was confirmed by sequence analysis of the rDNA internal transcribed spacers (ITS; GenBank Accession Nos. KC248202 and KC248201), and of the ß-tubulin (ß-tub; KC248404 and KC248403) and translation elongation factor 1-α (EF1-α; KC248206 and KC248205) genes. Comparative sequence analysis against the NCBI nucleotide database showed a high degree of identity with reference sequences of P. nicotianae (ITS, 99%; ß-tub, 99%; EF1-α, 100%) (2). A pathogenicity test with a representative isolate of P. nicotianae was performed on 10-week-old healthy vinca seedlings (n = 10). An aliquot of 10 ml of a zoosporic suspension (104 zoospores/ml) was sprayed onto the seedlings' leaves. An equal number of non-inoculated control seedlings were sprayed with sterile distilled water. Seedlings were maintained in a moist chamber at 25°C with 80 to 90% relative humidity and watered as needed with sterile water. Inoculated plants showed initial symptoms of foliar blight after 4 days, whereas control plants remained healthy. Ten days after inoculation, inoculated plants showed severe wilting. P. nicotianae was reisolated only from inoculated plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. nicotianae attacking annual vinca in northwestern Mexico. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) L. P. Kroon et al. Fungal Genet Biol. 41:766, 2004. (3) F. N. Martin et al. Plant Dis. 96:1080, 2013.

HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset.

BACKGROUND: T cells from HIV+ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8+ CD28- T cells. In order to compare cytokine production from T cells from these two states, CD4+ and CD8+ T cells from HIV+ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8+ T cell subsets CD28+ and CD28- from the HIV+ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls. RESULTS: Flow cytometric analysis indicated that CD8+ T cells from both HIV+ and aged donors showed an increase of approximately 2-3 fold over controls in percentage of cells producing inflammatory cytokines IFN-gamma and TNF-alpha. Similar analysis also revealed that the production of interleukins-4,6 and 10, production was very low (1-2% of cells) and unchanged in these cells. Quantitative PCR also showed a substantial increase (4-5 fold) in IFN-gamma and TNF-alpha mRNA from HIV+ and aged CD8+ T cells, as did ELISA for secreted IFN-gamma and TNF-alpha (2.3-4 fold). Flow cytometric analysis showed that the CD8+ CD28- T cell subset accounts for approximately 80-86% of the IFN-gamma and TNF-alpha production from the CD8+ subset in the aged and HIV+ states. The CD4+ T cell, while not significantly changed in the HIV+ or aged states in terms of IFN-gamma production, showed a small but significant increase in TNF-alpha production in both states. CONCLUSIONS: Our data appear compatible with physiologic conditions existing in HIV+ and aged individuals, i.e. elevated serum levels and elevated CD8+ T cell production of IFN-gamma and TNF-alpha. Thus, the capacity for increased production of cytokines IFN-gamma and TNF-alpha in the aged individual by the dominant CD8+ CD28- subset may have a profound influence on the clinical state by aggravating inflammatory pathologies such as rheumatoid arthritis, and possibly Alzheimer's disease and Crohn's disease. In AIDS, these cytokines may contribute to wasting and cachexia. We theorize that the predominant phenotypic change to the cytotoxic CD8+ CD28- T cell subsets in both the HIV+ and the aged states may reflect a natural "endpoint" in CD8+ T cell differentiation induced after a lifetime of immune activity (toward viruses, etc) in the aged, and after a massive accelerated response to HIV in the HIV-positive individual.

Enhanced interferon-gamma by CD8+ CD28- lymphocytes from HIV+ patients.

Flow cytometric analysis of T cells from HIV+ and normal individuals activated for 15 hr showed that the percentage of cells producing interferon-gamma (INFgamma) was enhanced approximately threefold (39 compared to 14%) in the HIV+ CD8+ population. Activation modes, other than anti-CD3 with PMA, were ineffective, and in no case did the percentage of HIV+ CD4+ T cells show increased INFgamma production over controls. Enhanced INFgamma production was not induced by either anti-CD3 or PMA alone, or anti-CD3 or ConA with anti-CD28, or enhanced by N-acetylcysteine. In contrast to INFgamma production, the percentage of CD4+ T cells producing interleukin-2 (Il-2) greatly exceeded that of the CD8+ T cells. The results from flow cytometry analyses of HIV+ CD8+ T cells was supported by quantitative analysis of INFgamma mRNA (by PCR) and INFgamma secretion by ELISA. These methods showed a sixfold and three- to fivefold increase, respectively, on a per cell basis. As HIV infection progresses, as shown by loss of CD4+ T cells, the proportion of CD8+ CD28- T cells increases, and it is this T cell subset that is responsible for 80% or more of the enhanced INFgamma production. The enhanced INFgamma in HIV+ patients derives from two factors: the increase in CD8+ CD28- cells to 70% and the percentage producing INFgamma (60%, compared to 21% for CD8+ CD28+ cells). Our findings of a substantial increase in INFgamma production in HIV infection arising from the increased number of CD8+ CD28- T cells are compatible with clinical studies which show elevated INFgamma in HIV+ serum and INFgamma producing CD8+ T cells dominating HIV+ lymph nodes. We also found a significantly decreased proliferative response of the HIV+-derived CD8+ T cell fraction with coactivator anti-CD-28, in contrast to PMA (with anti-CD3), which is probably a reflection of the diminished population of CD8+ CD28+ T cells in HIV+ donors compared to normal donors (30.7 compared to 67.9%).

The proliferative response of HIV+ T-cells (CD4+ and CD8+) are severely suppressed via CD28 coactivation.

Several studies have suggested that regulation of expression of the costimulatory molecule CD28 on the T-cell surface may play an important role in AIDS pathogenesis. In a study of T-cells from HIV+ donors, we find that activation with anti-CD3 plus anti-CD28 results in a mitogenic response which was approximately 86% suppressed for both CD4+ and CD8+ T-cells when compared to normal control cells. With PMA costimulation (instead of anti-CD28), the anti-CD3 response was suppressed much less, by 64 and 61%, respectively. With Con A as opposed to CD3 stimulation, the degree of suppression was less with either coactivator but still more severe with CD28 than with PMA coactivation. It has been reported that the CD28 subset of CD8+ T-cells is diminished in HIV+ individuals and could account for these results. It is possible as well that the CD28 costimulatory pathway in the CD4+ T-cells particularly is altered due to intervention by the HIV. While our data do not differentiate between these two possibilities, it show that the immune status is compromised in the HIV+ individual not only in terms of number of CD4+ T-cells, but in their activation response as well.

The suppression of T cell function and NF(kappa)B expression by serine protease inhibitors is blocked by N-acetylcysteine.

Direct evidence that N-acetylcysteine (NAC) enhances the immune response of peripheral blood T cells at the level of NF(kappa)B is presented. In addition, NAC blocks the suppression of T cell mitogenesis and cytokine production by protease inhibitors such as N-tosylphenylalanine chloromethyl ketone (TPCK). The proliferative responses of purified CD4+ or CD8+ T cells are suppressed more strongly by TPCK when anti-CD28 rather than the phorbol ester PMA is used as the mitogenic coactivator. Cytokine (IL-2, IL-6, INF-gamma) production is inhibited 95-100% by concentrations of TPCK that totally suppress the mitogenesis of CD4+ or CD8+ cells. Using electrophoretic mobility shift assays, we find that TPCK virtually abolishes (to less than 1%) the levels of NF(kappa)B (but not Oct-1) found in nuclear and whole cell extracts of activated T cells. Strikingly, the immunosuppressive effects of TPCK are blocked when T cells are pretreated for 15 min with 5 mM NAC. NAC not only blocks the effect of TPCK but enhances mitogenesis and cytokine production (>2.5-fold in some cases) upon activation of unsuppressed T cells. Our data support the notion that NF(kappa)B and I(kappa)B proteases play obligate roles in T cell activation and mitogenesis, roles that are enhanced significantly by NAC.

Slow cluster formation of purified human or rhesus T cells requires protein kinase C and LFA-1.

Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74% of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26%. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more sensitive to staurosporin inhibition, and is not stimulated via the TCR receptor like the rapid adhesion process. We hypothesize that certain neuronal processes, induced by phorbol ester, and which also show a similar protein kinase C activation time course, may share mechanisms in common with cluster formation.

Sustained levels of ascorbic acid are toxic and immunosuppressive for human T cells.

Ascorbate (vitamin C) can protect from oxidative damage to DNA and lipids that may lead to aging, cancer, and other dysfunctions. However, we find that purified human T cells deteriorate if maintained in ascorbate in culture for 18 hrs. or more; viability and Il-2 synthesis are over 90% curtailed by ascorbate at 50 micrograms/ml. T cell proliferation and adhesion are severely suppressed at 10-25 micrograms/ml. Dihydro-ascorbate was much less toxic or suppressive. The suppressive effect of ascorbate appears irreversible, since removal of ascorbate after 18 hrs. did not restore the mitogenic response. Although moderate dietary levels of ascorbate often reach 250-1000 mg or more daily and appear beneficial, our data caution against sustained megadoses of ascorbate for treatment of patients with AIDS and cancer.

Suppressed proliferative response and interleukin-2 production in hispanic HIV+ and AIDS T-cell subsets.

We find that interleukin-2 (IL-2) production is severely depressed (80-90%) in AIDS T-cells (CD4+ or CD8+) stimulated with anti-CD3 or Con A together with phorbol ester (PMA) or anti-CD28 coactivation. Likewise, the proliferative response of CD4+ T-cells was suppressed, from a mean of 24.6% (HIV+) to 59.1% (AIDS) for PMA with activators OKT3 (anti-CD3), Con A, enterotoxin B or pokeweed mitogen, and 20.2% (HIV+) to 77.8% (AIDS) with anti-CD28 co-activation. Similar degrees of suppression were found with the CD8+ T-cells except for a much greater suppression at the HIV+ stage with anti-CD28 (57.7%), approximately 2.5 times higher than for PMA coactivation. However, when proliferation was induced by the two coactivators combined (PMA plus anti-CD28), much less suppression was observed: 8.5% (HIV+) to 19.0% (AIDS) for CD4+ cells and 8.2% to 26.5%, respectively, for CD8+ cells. The data suggest that during HIV infection the CD28 pathway becomes most defective, but can be bypassed to some extent by the less-impaired PMA pathway. The IL-2 (+PMA) signal in HIV+ and AIDS cells was also significantly less suppressed suggesting that the disregulation in HIV infection is more prominent prior to the IL-2 stage of the mitogenic pathway. It is remarkable that the CD4+ and CD8+ T-cells at both the HIV+ and AIDS stages generally show the same degree of suppression with all the various activators and coactivators used.(ABSTRACT TRUNCATED AT 250 WORDS)

N-acetylcysteine (NAC) enhances interleukin-2 but suppresses interleukin-4 secretion from normal and HIV+ CD4+ T-cells.

We find that purified CD4+ T cells from 30 HIV+ individuals have a suppressed Interleukin-4 (IL-4) production compared to normal controls regardless of activator (anti-CD3 or Con A) or co-activator [phorbol ester (PMA or anti-CD28)], generally by 2-4 fold. In every case, the cells producing IL-4 respond more strongly to anti-CD28 co-activation than to PMA, ie, 1150 pg/ml compared to 2070 pg/ml for controls and 398 pg/ml compared to 1250 pg/ml for HIV+ cells, respectively. In contrast, anti-CD3 with PMA gives a more vigorous IL-2 response than with anti-CD28, ie, 37.3 ng/ml compared to 12.3 ng/ml for controls and 28.5 ng/ml versus 15.1 ng/ml for HIV+ cells, respectively. These data are not compatible with the TH1/TH2 switch hypothesis since IL-4 production is decreased, not increased for CD4+ HIV+ T-cells and while IL-2 production is decreased with PMA, it is not decreased significantly with anti-CD28. Interestingly, 5 mM N-acetylcysteine (NAC) acts as an immunoenhancer; mitogenesis was enhanced 2 fold or more in general for control and HIV+ CD4+ T-cells and IL-2 production was enhanced 2-3 fold for anti-CD3 (with PMA or anti-CD28) for both controls and HIV+ CD4+ cells. However, NAC suppressed IL-4 production induced by anti-CD3 and anti-CD28 in both control and HIV+ CD4+ T cells. In the other cases, it produced in general no significant change.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-CD2-induced tyrosine phosphorylation of T cell polypeptides is independent of the PMA-induced modification of p56lck.

The molecular basis for T cell activation involves the phosphorylation of of polypeptides at both serines and tyrosines. We find that with human peripheral T cells the serine phosphorylation of p56lck is independent of the more rapid tyrosine phosphorylation of other polypeptides via stimulation of the CD2 receptor with anti-CD2 (anti-T11(2) and anti-T11(3) mAb's). Triton X-100 soluble polypeptides were analyzed by Western blotting with the subsequent immunodetection by anti-phosphotyrosine or anti-lck antibodies. While polypeptides from resting T cells showed very low levels of endogenous tyrosine phosphorylation, incubation with anti-CD2 for periods as short as 30 sec resulted in the tyrosine phosphorylation of a 75-kDa polypeptide (p75). Polypeptide bands were also observed at 27 and 54 kDa, but these were artifacts from the reaction of anti-CD2 with the horse anti-mouse secondary antibody used in our detection system. Preincubation of T cells with phenylarsine oxide amplified the anti-CD2-induced tyrosine phosphorylation of the p75 and revealed additional phosphotyrosine polypeptides of 120, 100, and 33 kDa. The mitogenic combination of phorbol 12-myristate 13-acetate (PMA) with anti-CD2 changed neither the intensity nor the pattern of the tyrosine phosphorylation observed with anti-CD2 alone. The tyrosine phosphorylation of the p75 was not induced by concanavalin A (Con A) or PMA. While PMA alone failed to stimulate tyrosine phosphorylation above resting levels, PMA induced the nearly complete conversion of p56lck into p60lck (the lck-shift) at 30 min; no lck-shift was observed at 30 and 90 sec. Neither anti-CD2 nor Con A induced the lck-shift. Whereas PMA with either anti-CD2 or Con A was required for mitogenesis, only anti-CD2 led to tyrosine phosphorylation, and only PMA induced the lck-shift.

N-acetylcysteine enhances T cell functions and T cell growth in culture.

N-Acetylcysteine (NAC) is highly nontoxic for peripheral blood T cells and immunostimulatory enhancing T cell functions such as mitogenesis, interleukin-2 (IL-2) production, and growth in culture. NAC has been proposed for the treatment of AIDS based on its inhibition of human immunodeficiency virus (HIV) replication in cultured cells. Therefore its effect on normal T cells from 10 young donors and one elderly donor has been investigated as a prelude to clinical consideration. T cell function was evaluated in the presence and absence of accessory cells. With concanavalin A and anti-CD3 activation, NAC enhanced mitogenesis by approximately 2- to 2.5-fold at 5-10 mM. Mitogenesis of purified T cells with anti-CD2 was not affected by NAC; in the presence of accessory cells, NAC enhanced mitogenesis by approximately 2-fold at 1-10 mM. Importantly, NAC levels above 10 mM completely inhibited activation of peripheral blood mononuclear cells by anti-CD2. IL-2 secreted by T cells was also enhanced by NAC, approximately 1.5-fold, but IL-2 secreted by cells from old donors was enhanced by 3-fold. In cultures of peripheral blood T cells, NAC (10 mM) stimulated growth by at least 4- to 6-fold after two passages. These results show that NAC, nontoxic even at 20 mM, is an effective enhancer of T cell function and a remarkable enhancer of growth. Results from other laboratories show that NAC, which increases glutathione levels, suppresses HIV replication presumably via suppression of the activation of transcriptional factor NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)