Exposure to HIV-protease inhibitors selects for increased expression of P-glycoprotein (ABCB1) in Kaposi's sarcoma cells.

BACKGROUND: Given that HIV-protease inhibitors (HIV-PIs) are substrates/inhibitors of the multidrug transporter ABCB1, can induce ABCB1 expression, and are used in combination with doxorubicin for AIDS-Kaposi's Sarcoma (KS) treatment, the role that ABCB1 plays in mediating multidrug resistance (MDR) in a fully transformed KS cell line (SLK) was explored. METHODS: The KS cells were exposed to both acute and chronic treatments of physiological concentrations of different HIV-PIs (indinavir, nelfinavir, atazanavir, ritonavir, or lopinavir), alone or together with doxorubicin. The ABCB1 mRNA and protein expression levels were then assessed by qRT-PCR and western blotting, flow cytometry, and immunofluorescence. RESULTS: Chronic treatment of SLK cells with one of the five HIV-PIs alone or together resulted in increased resistance to doxorubicin. Co-treatment with one of the HIV-PIs in combination with doxorubicin resulted in a synergistic increase in resistance to doxorubicin, and the degree of resistance was found to correlate with the expression of ABCB1. The SLK cells were also revealed to be cross-resistant to the structurally unrelated drug paclitaxel. CONCLUSION: These studies suggest that ABCB1 is primarily responsible for mediating MDR in SLK cells selected with either HIV-PIs alone or in combination with doxorubicin. Therefore, the roles that ABCB1 and drug cocktails play in mediating MDR in KS in vivo should be evaluated.

Cyclophosphamide Effect on the Epithelial Covering of Rats Fetuse's Tongue: A Morphometric Study / Efectos de la Ciclofosfamida en el Epitelio de Revestimiento de la Lengua de Fetos de Rata: Un Estudio Morfométrico

The present study has the aim to describe morphologically, morphometrically and stereologically some changes in the tongue epithelium of rat fetuses under the effect of cyclosphosphamide. This drug was used in a single 6mg/kg according to the body weight in the tenth ­ pregnancy day while the control group received equal dosage of saline solution by the same duct and in the same day of pregnancy. The fetuses were plunged into fixed solution of Bouin for five days and the heads of then were divided and blocked in paraffin. Histological sections of six-micrometer thickness were performed and then colored with hematoxilin and eosin. It was used morphometrical and stereological analysis after the morphological study. The data were submitted under the statistical non-parametric test of Mann-Whitney. The results showed that the cyclosposphamide usage effect during the rat pregnancy caused a delayed in the embryo fetal growing, a reduction in the placental weight as wells as in the umbilicus cord length. The covering epithelia in the tongue of the treated group showed some changes since there was an epithelial hyperplasia associated with a cell hypotrophy. The fetuses in this group were more premature than the ones in the control.

El estudio tiene como objetivo describir morfológica, morfométrica y estereológicamente algunos cambios en el epitelio de la lengua de fetos de rata bajo los efectos de la ciclofosfamida. Esta droga fue usada en una muestra única de 6mg/kg de acuerdo al peso del cuerpo, en el décimo día de preñez, mientras el grupo control recibió igual dosis de una solución salina, por la misma vía, en los mismos días de preñez. Los fetos fueron sumergidos en solución fijadora de Bouin, por cinco días y las cabezas de ellos fueron cortadas y colocadas en bloques de parafina. Secciones histológicas de 6 µm fueron teñidas con hematoxilina-eosina. Después del estudio morfológico se realizaron análisis morfométricos y estereológicos. Se utilizó el test estadístico no paramétrico de Mann-Whitney. Los resultados mostraron que el uso de la ciclofosfamida provoca efectos durante la preñez de la rata, causando un retardo en el crecimiento de los embriones, una reducción en el peso de las placentas como también, en la longitud del funículo umbilical. El epitelio de revestimiento de la lengua del grupo tratado, mostró algunos cambios desde una hiperplasia epitelial asociada con una hipotrofia celular. Los fetos del grupo tratado fueron más prematuros que los del grupo control.

HIV-protease inhibitors contribute to P-glycoprotein efflux function defect in peripheral blood lymphocytes from HIV-positive patients receiving HAART.

P-glycoprotein (P-gp) has been found expressed in normal human cells, such as bone marrow and peripheral blood cells. The aim of this study was to investigate whether HIV-protease inhibitors (HIV-PIs) interact with P-gp efflux function in normal human peripheral blood lymphocytes (PBLs) and CD34+ progenitor cells. Moreover, we analyzed the in vivo effect of HIV-PIs on P-gp function in PBLs from HIV-infected patients receiving highly active antiretroviral therapy (HAART). We found that HIV-PIs (i.e., ritonavir, saquinavir, nelfinavir and indinavir) interfere with P-gp function in normal PBLs as demonstrated by the reduced efflux of rhodamine 123 (Rh123). This effect was dose-dependent and suggested the following hierarchy: ritonavir > saquinavir > nelfinavir > indinavir. We further analyzed the effect of HIV-PIs on the P-gp function in specific PBLs subsets. Our results show an HIV-PI-induced inhibition of P-gp function in CD4+ and CD8+ T cell subsets, mostly caused by the effect on the naive compartment of both CD4+ and CD8+ T cells. The same inhibitory effect was found in CD34+ hematopoietic progenitor cells. With respect to the in vivo evaluation of P-gp function in PBLs from HIV-infected patients, we found reduced levels of Rh123 efflux that reached the lowest value in AIDS patients receiving HAART. We concluded that HIV-PIs interfere with P-gp function in major cellular targets for HIV infection, such as CD4+ T cells and CD34+ progenitor cells. This ability may contribute to P-gp efflux function defect found in HIV-infected patients and suggests that drug interaction studies are crucial to an overall understanding of the effects of this important group of drugs.

Decreased function of Fas in patients displaying delayed progression of HIV-induced immune deficiency.

INTRODUCTION: In acquired immune deficiency syndrome patients, apoptosis of uninfected lymphocytes may contribute to development of immune deficiency. This process may involve recruitment of Fas by human immunodeficiency virus products. In line with this possibility, the viral envelope glycoprotein gp120 does not induce death of T cells from subjects with the autoimmune/lymphoproliferative syndrome displaying defective Fas function. This study evaluates the possibility that Fas function defects delay progression of HIV-induced immune deficiency. MATERIALS AND METHODS: The susceptibility to Fas-induced cell death was assessed on T cells from 18 'long-term non-progressor', four 'non-progressor', four 'progressor' asymptomatic HIV-1-infected, and nine AIDS patients using anti-Fas monoclonal antibodies. RESULTS: Fas-induced cell death was significantly lower in long-term non-progressors and non-progressors than in normal controls, progressors, and AIDS. The single-patient data showed that 9/18 long-term non-progressors and 3/4 non-progressors, but no progressors or AIDS were resistant to Fas. Analysis of the uninfected parents of two long-term non-progressors displaying decreased Fas-function showed that the mother of one of them and the father of the other displayed the same Fas function defect as their children. Fusion of T cells from Fas-resistant individuals with a Fas-sensitive cell line gave rise to Fas-resistant hybrid lines not carrying HIV, which suggests that the resistant phenotype is due to molecules exerting a dominant negative effect on a normal Fas system. CONCLUSION: These data suggest that Fas-resistance in long-term non-progressors may be due to inherited alterations of the Fas signaling pathway and may be a novel factor in delayed progression.

Expression of the novel T cell activation molecule hpH4 in HIV-infected patients: correlation with disease status.

We have described hpH4, a surface glycoprotein selectively expressed by activated T cells and mature thymocytes and displaying weak lateral association with CD4. The hpH4 expression pattern and biochemical features, together with analysis of its tryptic digest by peptide mass searching using MALDI-MS, suggested that it is a novel molecule. The aim of this work was to evaluate the peripheral blood T cell expression of hpH4 in HIV-infected patients and the interplay between HIV gp120 and hpH4, since both molecules interact with CD4. hpH4 expression during HIV-1 infection was evaluated by assessing 55 patients at various disease stages and following up 3 patients with primary infection and 3 patients with AIDS. hpH4 expression displayed a peak in the early phase of primary infection, dropped to control levels in the asymptomatic phase, and was newly expressed, at low levels, as AIDS developed. The expression kinetics were different than those shown by HLA-DR, CD25, and CD38. The most striking findings were the transient hpH4 expression peak displayed in the earliest stage, which was unique for hpH4. Incubation of T cells from normal donors with HIV gp120 induced transient hpH4 expression in resting CD4+ T cells and potentiated the hpH4 lateral association with CD4 in activated T cells. Moreover, hpH4 triggering inhibited gp120-induced death of CD4+ cells. Therefore, H4 expression may be a response to avoid apoptosis induced by HIV products.

Non-AIDS-defining neoplasms and HIV infection.

The purpose of this study was to characterise the epidemiological and the clinical features of non-AIDS-defining neoplasms (NAN) in HIV-infected subjects in the era of highly active antiretroviral therapy (HAART). A retrospective cohort of 4,041 subjects was established. Patients were recruited from January 1989 to December 1998. We observed 51 NAN over the study period. The overall incidence rate was 0.21 per 100 person-years (PY) and it remained stable, also after the introduction, in 1996 of HAART. Moreover, stratifying according to the type of neoplasms there was no statistically significant difference in the incidence of NAN over the study period. While the epidemiological features of NAN generally was not different from that observed in immunocompetent individuals, the neoplasms had a more aggressive clinical course and a poor prognosis. Survival analysis showed that the presence of NAN significantly reduced the survival of patients with AIDS (P=0.01; OR=0.62; 95% CI=0.47-0.96) compared with matched controls. The overall mortality-rate was 63% with an incidence rate of 0.13 per 100 PY. Although the incidence rate of NAN is not of great magnitude, as the number of HIV-infected individuals continues to increase and their survival improves, the number of HIV-infected subjects with NAN might consequently increase as well as the related morbidity and mortality.

Induction of CD69 antigen on normal CD4+ and CD8+ lymphocyte subsets and its relationship with the phenotype of responding T-cells.

We evaluated phenotype and apoptotic status of normal CD4+CD69+ and CD8+CD69+ peripheral blood T-lymphocytes after short-term challenge with escalating concentrations of phytohemagglutinin (PHA). The frequency of CD69-coexpressing CD4+ and CD8+ T-cells and CD69 staining intensity increased following T-cell mitogenic stimulation; these changes were proportional to PHA concentration in culture medium. A considerable fraction of lymphocytes underwent blast transformation, displaying increased forward and side scatter signals. Interestingly enough, PHA-responsive T-cells exhibited a predominantly CD25negCD38negTCRalphabetapos phenotype; APO-1/Fas antigen (CD95) could be detected on a minority of activated CD69+ T-cells. A considerable proportion of CD69+ lymphocytes expressed intracellular perforin; in addition, an average 16+/-6% CD69+ T-lymphocytes were apoptotic after 4 h of stimulation, as evaluated by 7-amino-actinomycin-D staining and by annexin-V binding. CD69+ activated lymphocytes comprise phenotypically heterogeneous cell subpopulations potentially devoted to diverse immunological functions, i.e., proliferation, apoptosis, or cell cytotoxicity; moreover, our findings indicate that CD69 expression is proportional to the intensity of the activating stimulus and that the capacity to upregulate CD69 antigen following short-term mitogenic challenge may be restricted to unactivated CD38negCD25negTCRalphabetapos T-lymphocytes.

Serum of healthy donors receiving granulocyte colony-stimulating factor induces T cell unresponsiveness.

The effects of serum from healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF) (G-serum) on blast transformation, expression of activation-related antigens, secretion of interleukin (IL)-2, and proliferation were evaluated in allogeneic lymphocytes stimulated with phytohemagglutinin. Escalating concentrations of G-serum induced 27%, 47%, and 70% suppression of lymphocyte proliferation; interestingly, CD4+ and CD8+ cells underwent blast transformation and up regulated early (CD69) and late (CD25, HLA-DR, and CD71) activation-related antigens. Negligible fractions of apoptotic cells were found after mitogenic challenge, suggesting that the strongly diminished proliferation was not attributable to extensive activation-induced programmed cell death of responding T cells. The levels of IL-2 in cultures containing G-serum were comparable to those in cultures performed without G-serum; however, high concentrations of exogenous IL-2 restored lymphocyte mitogenesis regardless of G-serum concentration. These findings--cell enlargement, upregulation of activation-related antigens, inability to proliferate after mitogenic stimulus, and restoration of cell division by exogenous IL-2--resembled those associated with "partial activation" of lymphocytes, a fundamental control mechanism of tolerance induction in T cell clones. Soluble immunoregulatory mediators infused with allogeneic hematopoietic progenitor products collected after rhG-CSF administration could induce T cell unresponsiveness in vivo, thus preventing clonal expansion and amplification of immune responses, and could account for the unexpectedly reduced incidence and severity of graft vs. host disease compared with allogeneic marrow infusion.

Intracellular expression of P-170 glycoprotein in peripheral blood mononuclear cell subsets from healthy donors and HIV-infected patients.

BACKGROUND AND OBJECTIVE: P-glycoprotein (P-gp) is a transmembrane efflux pump that actively extrude a variety of unrelated drugs from cancer cells, leading to the so-called multidrug resistance (MDR) phenomenon. However, P-gp has also been found in normal bone marrow and peripheral blood mononuclear cells (PBMC). Recently, the presence of P-glycoprotein in PBMC from human immunodeficiency virus (HIV)-infected patients has also been investigated and a phenotype-associated P-gp expression has been detected. DESIGN AND METHODS: A total of thirty-eight HIV-1 positive patients with a mean age of 34 years (range, 24-41 years) were studied after an informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll/Hypaque and P-glycoprotein expression was investigated on lymphocyte population by single and double-color immunofluorescence techniques. We investigated: i) both surface and intracellular expression of the P-gp molecule in different PBMC subsets, ii) P-gp expression modifications occurring during HIV infection, and iii) the effect of HIV-gp120 on the expression of P-gp by T lymphocyte subsets from healthy donors. RESULTS: Our experimental findings indicate that: a) P-gp glycoprotein can be detected on an intracellular level in different PBMC subpopulations (mainly CD8+ T lymphocytes, CD16+ NK cells and CD14+ monocytes); b) this intracellular expression is decreased in specific PBMC subsets (i.e. T-CD8+ and NK-CD16+) from HIV-infected patients and c) a rearrangement was obtained when CD4+ and CD8+ lymphocytes from healthy donors were exposed in vitro to the HIV-binding glycoprotein gp120. INTERPRETATION AND CONCLUSIONS: Our results indicate that P-gp glycoprotein can also be expressed intracellularly and can be rearranged in PBMC subsets from HIV-infected patients.

A new, striking morphological alteration of P-glycoprotein expression in NK cells from AIDS patients.

Natural killer cells from healthy donors express P-glycoprotein on their surface. This molecule is rearranged during the process of cell-mediated cytotoxicity and it appears to be clustered in the cell-to-cell contact regions. By contrast, in HIV-infected subjects this rearrangement is hindered. These results seem to be associated with cytoskeleton network alterations of the cell-mediated killing process occurring in AIDS patients and can contribute to the comprehension of the mechanisms of the natural killer cell deficiency found in these patients.

Flow cytometric detection of perforin in normal human lymphocyte subpopulations defined by expression of activation/differentiation antigens.

We investigated with three-color flow cytometry the expression of perforin (Pf) in normal human lymphocyte subpopulations identified by means of activation and differentiation-related antigens. Interestingly, Pf could be detected in a substantial subset (13 +/- 2%) of memory CD4+ CD45RO+ cells, on relevant percentages of memory (CD45RO+) and naive (CD45RA+) CD8+ cells and on virtually all CD3- CD16+, CD3- CD56+ and NKB1+ natural killer cells, as expected. The analysis of fluorescence intensity showed higher levels of Pf expression on CD8dim and NK cells compared to CD8bright and CD4+ lymphocytes, Pf and CD69, HLA-DR, CD95 and CD25 activation/differentiation-related antigens were never co-expressed. On average, 15 +/- 3% of CD3+ CD28+ cells were found to be Pf+, in line with a previously activated or memory cell type. Comparable percentages of CD8+ CD11b- (cytotoxic) and CD8+ CD11b+ (suppressor) T cells were Pf+. Multiparameter flow cytometry is a powerful tool to detect minute fractions of Pf-expressing cells in heterogeneous populations.

Bilateral parotid sialolithiasis in a patient with acquired immunodeficiency syndrome and immunoglobulin G multiple myeloma.

Bilateral multiple parotid calculi, which are uncommonly diagnosed in the normal population, have never been reported in patients infected with human immunodeficiency virus. Herein we report a case of bilateral parotid sialolithiasis in a patient who had acquired immunodeficiency syndrome and was affected by multiple myeloma. The possible etiopathogenesis in view of the alterations of immunity, oral pH, and salivary composition that are observed in multiple myeloma and in human immunodeficiency virus infection are discussed.

Gastric cryptosporidiosis complicating HIV infection: case report and review of the literature.

Gastric cryptosporidiosis is rarely reported despite the fact that Cryptosporidium has been recognized with increasing frequency as an aetiological agent of severe chronic diarrhoeal enteritis in patients with human immunodeficiency virus (HIV) infection. We report a case of gastric cryptosporidiosis that occurred in an AIDS patient. Fifteen previously reported cases of gastric cryptosporidiosis are also reviewed. The review of all cases reported in the literature, including our own, showed that 12 patients had digestive symptoms. In 14 out of 16 patients the diagnosis was achieved by biopsy of the gastric mucosa rather than by examination of the stools. Gastric localization of Cryptosporidium is a rare and severe manifestation of intestinal cryptosporidiosis in patients with AIDS, and no single drug regimen has yet been found to be effective in eradicating this organism.

Central venous catheter-related sepsis in a cohort of 366 hospitalised patients.

Five hundred two central venous catheters inserted in 366 patients were evaluated prospectively over a one-year period to determine the frequency and risk factors associated with catheter-related sepsis. For study purposes, in cases in which catheter infection was suspected but the initial blood cultures were negative, the catheters were replaced by guidewire technique; otherwise, the catheters were routinely changed after 21 days by guidewire technique. A catheter-related infection was suspected in 190 cases (190/502, 38%). A diagnosis of catheter-related sepsis was established in 50 patients, which represents 10% of the total number of lines (502). Over a total of 6428 days of catheter use, the infection rate was 0.8 cases of sepsis per 100 catheter-days. Staphylococcus epidermidis, Staphylococcus aureus, and Candida spp. were the most frequently isolated aetiological agents of sepsis. On univariate analysis, six variables affecting the rate of catheter-related sepsis were identified: neutropenia for more than eight days (p < 0.001); AIDS (p < 0.001); haematological malignancy (p < 0.001); administration of total parenteral nutrition (p = 0.001); duration of site use (p = 0.04); and high APACHE II score (p = 0.04). The logistic regression analysis revealed that AIDS and haematological malignancies were independent risk factors of catheter-related sepsis. Catheter replacement over a guidewire was no more likely to be associated with sepsis than was percutaneous catheter insertion. In conclusion, although the incidence of established catheter infection is much lower than the incidence of suspected infection, in most cases of suspected infection it is wise to change the catheter with the guidewire technique and wait for culture of the tip, rather than to remove the catheter immediately. Such a policy may help reduce the number of unnecessary catheter removals.