Zooming in on size distribution patterns underlying species coexistence in Baltic Sea phytoplankton.

Scale is a key to determining which processes drive community structure. We analyse size distributions of phytoplankton to determine time scales at which we can observe either fixed environmental characteristics underlying communities structure or competition-driven size distributions. Using multiple statistical tests, we characterise size distributions of phytoplankton from 20-year time series in two sites of the Baltic Sea. At large temporal scales (5-20 years), size distributions are unimodal, indicating that fundamental barriers to existence are here subtler than in other systems. Frequency distributions of the average size of the species weighted by biovolume are multimodal over large time scales, although this is the product of often unimodal short-term (<1 year) patterns. Our study represents a much-needed structured, high-resolution analysis of phytoplankton size distributions, revealing that short-term analyses are necessary to determine if, and how, competition shapes them. Our results provide a stepping-stone on which to further investigate the intricacies of competition and coexistence.

Increased cell division but not thymic dysfunction rapidly affects the T-cell receptor excision circle content of the naive T cell population in HIV-1 infection.

Recent thymic emigrants can be identified by T cell receptor excision circles (TRECs) formed during T-cell receptor rearrangement. Decreasing numbers of TRECs have been observed with aging and in human immunodeficiency virus (HIV)-1 infected individuals, suggesting thymic impairment. Here, we show that in healthy individuals, declining thymic output will affect the TREC content only when accompanied by naive T-cell division. The rapid decline in TRECs observed during HIV-1 infection and the increase following HAART are better explained not by thymic impairment, but by changes in peripheral T-cell division rates. Our data indicate that TREC content in healthy individuals is only indirectly related to thymic output, and in HIV-1 infection is mainly affected by immune activation.

Limited CD4+ T-cell renewal in early HIV-1 infection: effect of highly active antiretroviral therapy.

We show that the fraction of proliferating CD4+ lymphocytes is similar in HIV-infected subjects in the early stage of disease and in HIV-negative subjects, whereas the fraction of proliferating CD8+ lymphocytes is increased 6.8-fold in HIV-infected subjects. After initiation of antiviral therapy, there is a late increase in proliferating CD4+ T cells associated with the restoration of CD4+ T-cell counts. These results provide strong support for the idea of limited CD4+ T-cell renewal in the early stage of HIV infection and indicate that after effective suppression of virus replication, the mechanisms of CD4+ T-cell production are still functional in early HIV infection.

Refined characterization and reference values of the pediatric T- and B-cell compartments.

Work in the past years has led to a refined phenotypical description of functionally distinct T- and B-cell subsets. Since both lymphocyte compartments are established and undergo dramatic changes during childhood, redefined pediatric reference values of both compartments are needed. In a cohort of 145 healthy children, aged 0-18 years, the relative and absolute numbers of the various T- and B-cell subsets were determined. In addition, we found that besides thymic output, naive (CD27(+)CD45RO(-)) T-cell proliferation contributed significantly to the establishment of the naive T-cell compartment. At birth, regulatory (CD25(+)CD127(-)CD4(+)) T cells (Tregs) mainly had a naive (CD27(+)CD45RO(-)) phenotype whereas 'memory or effector-like' (CD45RO(+)) Tregs accumulated slowly during childhood. Besides the CD27(+)IgM(+)IgD(+) memory B-cell population, the recently identified CD27(-)IgG(+) and CD27(-)IgA(+) memory B-cell populations were already present at birth. These data provide reference values of the T- and B-cell compartments during childhood for studies of immunological disorders or immune reconstitution in children.

Programmed death of T cells in HIV-1 infection.

In human immunodeficiency virus (HIV) infection, functional defects and deletion of antigen-reactive T cells are more frequent than can be explained by direct viral infection. On culturing, both CD4+ and CD8+ T cells from asymptomatic HIV-infected individuals died as a result of programmed cell death (apoptosis). Apoptosis was enhanced by activation with CD3 antibodies. Programmed cell death, associated with impaired T cell reactivity, may thus be responsible for the deletion of reactive T cells that contributes to HIV-induced immunodeficiency.

T cell telomere length in HIV-1 infection: no evidence for increased CD4+ T cell turnover.

Progression to acquired immunodeficiency syndrome (AIDS) has been related to exhaustion of the regenerative capacity of the immune system resulting from high T cell turnover. Analysis of telomeric terminal restriction fragment (TRF) length, a marker for cellular replicative history, showed that CD8(+) T cell TRF length decreased but CD4(+) T cell TRF length was stable during the course of human immunodeficiency virus type-1 (HIV-1) infection, which was not explained by differential telomerase activity. This observation provides evidence that turnover in the course of HIV-1 infection can be increased considerably in CD8(+) T cells, but not in CD4(+) T cells. These results are compatible with CD4(+) T cell decline in HIV-1 infection caused by interference with cell renewal.

Quantitative analysis of CD4+ T cell function in the course of human immunodeficiency virus infection. Gradual decline of both naive and memory alloreactive T cells.

Early in human immunodeficiency virus (HIV) infection CD4+ and CD8+ T cells are qualitatively affected. Loss of responses to recall antigen precedes impaired responses to allogeneic MHC and mitogens. The selective quantitative loss of memory T cells in early infection, only partially explains the observed defects. We investigated whether functional loss of T cells is preferentially observed for memory T cells or whether both naive and memory T cell subsets are affected in the course of HIV infection. We studied the proliferative response of CD4+ T cells from HIV-infected individuals to alloantigens, to which normally both naive and memory T cells respond, by limiting dilution analysis. The decreased proliferative response to alloantigens in HIV-infected individuals was associated with a decreased precursor frequency of alloreactive cells. The frequency was decreased in both the CD45RA+ (naive) and the CD45RO+ (memory) subset of CD4+ T cells. Analysis of four individuals in the course of HIV infection revealed similar kinetics of the decline in function in both subsets. Although initially T cell defects may be accounted for by the selective quantitative loss of memory cells, in later stages of HIV infection the function of both CD45RA+ and CD45RO+ cells is affected.

Programmed death of T cells in human immunodeficiency virus infection. No correlation with progression to disease.

Programmed death of T cells has been proposed as one of the mechanisms by which HIV affects immune functions in stages of infection where the number of infected cells is low. Indeed, in HIV-infected individuals both CD4+ and CD8+ T cells are primed for programmed cell death, which can be enhanced by polyclonal stimulation. Here, we investigated programmed death of T cells in all stages of HIV infection, including acute infection. In individuals with primary infection the number of T cells dying due to apoptosis was much higher than in the asymptomatic phase of infection and paralleled increased numbers of CD8+ cells. In asymptomatic HIV-infected individuals, cells were dying in increased percentages compared with noninfected controls, although at much lower numbers than during acute infection. Death of T cells was not quantitatively correlated with CD4+ T cell numbers or appearance of more cytopathic, syncytium-inducing HIV variants. Analysis of the phenotype of cells undergoing apoptosis revealed that cell death was not confined to a specific T cell subset nor correlated with expression of certain T cell activation markers. Our results imply that the extent of programmed cell death of T cells in HIV infection does not correlate with progression to disease.

Immune restoration does not invariably occur following long-term HIV-1 suppression during antiretroviral therapy. INCAS Study Group.

BACKGROUND: Current antiretroviral treatment can induce significant and sustained virological and immunological responses in HIV-1-infected persons over at least the short- to mid-term. OBJECTIVES: In this study, long-term immune reconstitution was investigated during highly active antiretroviral therapy. METHODS: Patients enrolled in the INCAS study in The Netherlands were treated for 102 weeks (range 52-144 weeks) with nevirapine (NVP) + zidovudine (ZDV) (n = 9), didanosine (ddl) + ZDV (n = 10), or NVP + ddl + ZDV (n = 10). Memory and naïve CD4+ and CD8+ T cells were measured using CD45RA and CD27 monoclonal antibodies (mAb), T-cell function was assayed by CD3 + CD28 mAb stimulation, and plasma HIV-1 RNA load was measured by ultra-direct assay (cut-off < 20 copies/ml). RESULTS: Compared to both double combination regimens the triple combination regimen resulted in the most sustained increase in CD4+ T cells (change in CD4+, + 253 x 10(6) cells/l; standard error, 79 x 10(6) cells/l) and reduction of plasma HIV-1 RNA. In nine patients (31%) (ddl + ZDV, n = 2; NVP + ddl + ZDV, n = 7) plasma HIV-1 RNA levels remained below cut-off for at least 2 years. On average, these long-term virological responders demonstrated a significantly higher increase of naïve and memory CD4+ T cells (P = 0.01 and 0.02, respectively) as compared with patients with a virological failure, and showed improved T-cell function and normalization of the naïve; memory CD8+ T-cell ratio. However, individual virological success or failure did not predict the degree of immunological response. T-cell patterns were independent of baseline CD4+ T-cell count, T-cell function, HIV-1 RNA load or age. Low numbers of naïve CD4+ T cells at baseline resulted in modest long-term naïve T-cell recovery. CONCLUSIONS: Patients with prolonged undetectable plasma HIV-1 RNA levels during antiretroviral therapy do not invariably show immune restoration. Naïve T-cell recovery in the setting of complete viral suppression is a gradual process, similar to that reported for immune recovery in adults after chemotherapy and bone marrow transplantation.

Normal telomere lengths in naive and memory CD4+ T cells in HIV type 1 infection: a mathematical interpretation.

To study CD4+ T cell productivity during HIV-1 infection, CD4+ T cell telomere lengths were measured. Cross-sectional and longitudinal analysis of HIV-1-infected individuals with CD4+ T cells counts >300 cells/mm3 showed normal average telomeric restriction fragment (TRF) length and normal shortening rates of CD45RA+ naive and CD45RO+ memory CD4+ T cells. These TRF data were interpreted in terms of CD4+ T cell production by means of a mathematical model. This model resolves previous criticisms arguing that the normal TRF length of CD4+ T cells in HIV-1 clinical latency is due to the killing of dividing CD4+ T cells by the virus. Only an increased priming rate of naive CD4+ T cells to become memory cells may elongate the average TRF length of memory CD4+ T cells, and may therefore mask the shortening effect of increased turnover in the CD4+ memory T cell compartment. The data are more compatible with the notion that during HIV-1 clinical latency the turnover of CD4+ T cells is not markedly increased, however, and that HIV-related interference with renewal from progenitors plays a role in CD4+ T cell depletion. In such a "limited renewal" scenario disease progression is no longer a consequence of markedly increased CD4+ T cell production.

Functional B cell abnormalities in HIV type 1 infection: role of CD40L and CD70.

Early in HIV-1 infection, B cell responses to T cell-dependent antigens are impaired. In addition to the receptor-ligand pair CD40/CD40L, CD27/CD70 also appears to be involved in T cell-dependent B cell stimulation. We have shown that CD70+ B cells are the main producers of Ig when stimulated in a T cell-dependent manner, and that CD70 upregulation is dependent on interaction of CD40L on T cells with CD40 on B cells. We confirm here that B cells from HIV-infected individuals are impaired in T cell-dependent Ig production in vitro. This dysfunction could partly be restored by adding allogeneic T cells to the culture. In contrast, IgG production induced by CD40 MAb, IgM MAb, and IL-10 was in the normal range. In line with this, CD70 upregulation on B cells from HIV-infected individuals was impaired after stimulation in vitro by activated T cells but not after stimulation with CD40 MAb and IgM MAb. Furthermore, CD40L expression was decreased on CD4+ T cells after stimulation in vitro. Finally, CD70 expression on freshly isolated B cells from HIV-infected individuals was decreased, and low CD70 expression correlated with low IgG production after T cell-dependent stimulation. In conclusion, our data strongly suggest that impaired B cell responses to T cell-dependent Ag in HIV-1 infection are due to a defect in T cells.

Effects of HIV-1 Tat protein on human T cell proliferation.

In inducing immunodeficiency in human immunodeficiency virus (HIV)-1 infection, a role for the HIV-1 Tat protein has been suggested. In addition to effects on viral transcription, the protein has been ascribed immunosuppressive functions, which were investigated in this study. Proliferation of purified T cells to CD3 monoclonal antibodies immobilized to plastic plates was inhibited up to 70% by addition of 5 micrograms/ml of the Tat protein. This inhibitory effect, however, was not observed in the presence of accessory cells. Furthermore, no effect of Tat protein could be observed on T cell proliferative responses to recall antigen which most likely is related to the presence of accessory cells in the cultures. Taken together, our results do not imply an important role for immunosuppressive effects of Tat in induction of immunodeficiency as observed in HIV infection in vivo.

IL-12-induced IL-10 production by human T cells as a negative feedback for IL-12-induced immune responses.

IL-12 is an important initiator of cellular immune responses. This involves a positive feedback mechanism via IFN-gamma, which is abrogated when the pathogen that induces IL-12 production by the macrophage has been cleared. Here, we studied IL-10 as an additional negative regulator of IL-12-induced immune responses. Our results showed that upon stimulation with CD2 mAb, IL-12 was capable of inducing human T cells to produce IL-10. IL-12 was able to induce IL-10 production in primary T cells in the absence or the presence of accessory cells and in short-term cultures of established T cell clones. Moreover, T cell clones that had been cultured for longer periods in the presence of IL-12, when restimulated in the absence of IL-12, still produced high amounts of IL-10. Furthermore, we demonstrated that IL-10-mediated inhibition of T cell proliferation was dose dependent and depended on the time of addition of IL-10 and on the IL-12 concentration in culture. IL-10 has been identified as a dominant inhibitor of IL-12 production by APCs. The finding that IL-12 is capable of potently inducing its own inhibitor shows that the immune system is equipped with an intrinsic negative feedback mechanism that limits ongoing T cell activation. This indicates that the kinetics of T cell responses seem to be regulated by the ratio of IL-12 and IL-10 levels, which may gradually decline during the immune response.

Changes in cytokine secretion patterns of CD4+ T-cell clones in human immunodeficiency virus infection.

In addition to the loss of CD4+ T cells in later stages of human immunodeficiency virus (HIV) infection, functional defects of Th cells can already be observed in early infection. Decreased interleukin (IL)-2 and interferon (IFN)-gamma production by CD4+ T cells and diminished delayed type hypersensitivity reactions are indicative for impaired Th1 responses. We studied the cytokine secretion patterns of T-cell clones (TCC) generated by mitogenic stimulation of CD4+ memory T cells. Compared with TCC from HIV-negative controls, TCC isolated from HIV-infected individuals consistently showed increased IL-4 production, often paralleled by increased IL-5 and decreased IFN-gamma production. This resulted in a decreased percentage of Th1 clones with an increase in Th0 clones. To rule out the influence of interindividual differences, we studied two individuals from whom cells were available before and after infection with HIV. Indeed, an increase in Th2 cytokine secretion was observed after HIV-infection. Loss of Th1 and enhanced Th2 responses might further curtail cellular responses resulting in deficiency of cellular immunity in HIV infection.

Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation.

Antibodies against a novel adrenocorticotropic hormone-inducible cytochrome P450 (P450RAP), responsible for polycyclic aromatic hydrocarbon metabolism in rat adrenal microsomes (Otto, S., Bhattacharyya, K.K., and Jefcoate, C.R. (1992) Endocrinology 131, 3067-3076), identified a cDNA clone encoding a partial cytochrome P450 sequence from a rat adrenal cDNA library. Rescreening a second cDNA library yielded several clones up to 5.0 kilobases (kb) encoding a 1629-base pair open reading frame. The deduced amino acid sequence (543 residues) matched completely with five peptides cleaved from P450RAP. The amino acid sequence of P450RAP is 92% identical to a 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1B1, cloned from mouse C3H10T1/2 (10T1/2) embryo fibroblast cells (Savas, U., Bhattacharyya, K. K., Christou, M., Alexander, D.L., and Jefcoate, C. R. (1994) J. Biol. Chem. 269, 14905-14911), which shows nearly the same characteristics in polycyclic aromatic hydrocarbon metabolism. The available 5'- and 3'-noncoding regions show, respectively, 93 and 83% sequence identity. We conclude that P450RAP protein is encoded by a rat CYP1B1 gene orthologous to the mouse CYP1B1 gene. Alignment of rat CYP1B1 amino acid sequences with rat CYP1A1 (39% identical) indicated eight regions of high identity for each (60-78%), interspersed by extensive regions of less than 30% similarity. The CYP1B1 cDNAs hybridize a 5.2-kb mRNA in rat adrenals, consistent with the length of the longest clones and the mRNA recognized in 10T1/2 cells. CYP1B1 mRNA was elevated by a 2-day adrenocorticotropic hormone treatment but much less than CYP11A1 (cytochrome P450 side chain cleavage) mRNA (2-fold versus 4-fold). The lower levels of the 5.2-kb mRNA in other steroidogenic cells (ovary) was consistent with the amount of immunodetectable CYP1B1 protein and, unlike the adrenal, expression in the ovary was stimulated 5-fold by beta-naphthoflavone, an aryl hydrocarbon receptor agonist, in parallel with CYP1A1 induction. In several other tissues (liver > lung > uterus >> kidney), CYP1B1 mRNA and protein were constitutively undetectable but highly induced by beta-naphthoflavone, although at much lower levels than CYP1A1. Rat CYP1B1, therefore, exhibits regulation through hormonal signaling and the aryl hydrocarbon receptor in a cell-specific manner.

Evidence for intact costimulation via CD28 and CD27 molecules in hyporesponsive T cells from human immunodeficiency virus-infected individuals.

In the activation of T cells, the primary signal is antigen-specific and given through T cell receptor (TcR)/CD3 ligation. Furthermore, costimulatory molecules such as CD28 and CD27, provide an essential signal for activation through interaction with their ligands, present on the membrane of antigen-presenting cells. During asymptomatic human immunodeficiency virus (HIV)-1 infection, T cell function is progressively lost. Here, we investigated whether in the presence of impaired responses of T cells from HIV-infected individuals to signal one, costimulation through CD28 and CD27 after interaction with their natural ligands CD80 and CD70 is intact. T cell proliferative responses to signal one in combination with CD80 or CD70 were decreased in a large fraction of asymptomatically HIV-infected individuals. This was due to impaired responses of signal one but not to impaired responses to costimulation, since CD80 or CD70 did enhance signal one-mediated proliferative responses to a normal extent. Moreover, in individuals with proliferative responses to signal one that were decreased to 50% of normal T cell responses, costimulation even was increased compared to controls. Our results demonstrate that in HIV-infected individuals the response to costimulation is relatively preserved compared to responses to the first signal and point to the defect in T cells in HIV infection being primarily in the CD3/TcR-mediated pathway.

Single cell analysis of IL-4 and IFN-gamma production by T cells from HIV-infected individuals: decreased IFN-gamma in the presence of preserved IL-4 production.

The specific in vitro disturbance of capacities ascribed to Th1 cells in HIV-infected individuals suggests a switch from Th1 to Th2 lymphokine secretion. Indeed, when T cell clones are generated from HIV-infected individuals compared with controls, an increased percentage of Th0 clones is present upon HIV infection. We studied cytokine production in the supernatant of in vitro activated PBMC from a large group of HIV-infected patients at various stages of infection. IL-2, IFN-gamma, IL-4, IL-5, and IL-10 production all were decreased significantly, which does not support a switch to Th2 lymphokine secretion and is possibly due to the generalized impaired response of T cells from HIV-infected individuals to activation signals in vitro. Therefore, we investigated the capacity of single cells to produce a certain cytokine. Intracellular staining of IL-4- and IFN-gamma-producing cells revealed that T cells from HIV-infected individuals contained decreased numbers of IFN-gamma-producing cells, in the presence of normal percentages of cells with the capacity to produce IL-4. This resulted in significantly decreased IFN-gamma/IL-4 ratios in both CD4+ and CD8+ T cells. Thus, in agreement with previous findings in T cell clones, we conclude, from cytokine production upon stimulation of T cells in vitro, that there is a change in the cytokine balance to the Th2 side in HIV infection due to decreased Th1 and preserved Th2 cytokine production.

T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART).

In human immunodeficiency virus (HIV)-1 infection, highly increased T-cell turnover was proposed to cause exhaustion of lymphocyte production and consequently development of AIDS. Here, we investigated cell proliferation, as measured by expression of the Ki-67 nuclear antigen, in peripheral blood CD4(+) and CD8(+) lymphocyte subpopulations before and during highly active antiretroviral therapy (HAART). In untreated HIV-1 infection, both the percentage and number of Ki-67(+) CD4(+) and CD8(+) lymphocytes were significantly increased, compared with values obtained from healthy individuals. A more than 10-fold increase in the percentage of dividing naive CD4(+) T cells in the blood was found when the number of these cells were below 100 per microL. HAART induced an immediate decline in Ki-67 antigen expression, despite often very low CD4(+) T-cell numbers, arguing against increased proliferation being a homeostatic response. After approximately 24 weeks of HAART treatment, a transient increase in the number of proliferating cells was seen, but only in the CD4(+) CD27(+) memory pool. In the CD8(+) T-cell compartment, the number of dividing cells was elevated 20- to 25-fold. This increase was most notable in the CD27(+) CD 45RO(+) and CD27(-) CD45RO(+) memory CD8(+) T-cell pool, corresponding with the degree of expansion of these subsets. Reduction of plasma HIV-RNA load by HAART was accompanied by a decrease in numbers and percentages of dividing cells in all CD8(+) T-cell subsets. Taken together, our results indicate that peripheral T-cell proliferation is a consequence of generalized immune activation. (Blood. 2000;95:249-255)

The dominant source of CD4+ and CD8+ T-cell activation in HIV infection is antigenic stimulation.

To distinguish between antigenic stimulation and CD4+ T-cell homeostasis as the cause of T-cell hyperactivation in HIV infection, we studied T-cell activation in 47 patients before and during highly active antiretroviral therapy (HAART). We show that expression of human leukocyte antigen (HLA)-DR, CD38, and Ki67 on T cells decreased during HAART but remained elevated over normal values until week 48 of therapy. We confirm previous reports that T-cell activation correlates positively with plasma HIV RNA levels (suggesting antigenic stimulation), and negatively with CD4 count (suggesting CD4+ T-cell homeostasis). However, these correlations may be spurious, because misleading, due to the well-established negative correlation between CD4 count and plasma HIV RNA levels. To resolve this conflict, we computed partial correlation coefficients. Correcting for CD4 counts, we show that plasma HIV RNA levels contributed to T-cell hyperactivation. Correcting for plasma HIV RNA levels, we show that CD4+ T-cell depletion contributed to T-cell activation. Correcting for both, activation of CD4+ and CD8+ T cells remained positively correlated. Because this suggests that CD4+ and CD8+ T-cell activation is caused by a common additional factor, we conclude that antigenic stimulation by HIV or other (opportunistic) infections is the most parsimonious explanation for T-cell activation in HIV infection. Persistence of HIV antigens may explain why T-cell activation fails to revert to levels found in healthy individuals after 48 weeks of therapy.