Apoptosis of non-small-cell lung cancer cell lines after paclitaxel treatment involves the BH3-only proapoptotic protein Bim.

A significant variation in susceptibility to paclitaxel-mediated killing was observed among a panel of short-term cultured non-small-cell lung cancer (NSCLC) cell lines. Susceptibility to killing by paclitaxel correlated with expression of the BH3-only protein, Bim, but not with other members of Bcl-2 family. NSCLC cell lines with the highest level of Bim expression are most susceptible to apoptosis induction after paclitaxel treatment. Forced expression of Bim increased paclitaxel-mediated killing of cells expressing an undetectable level of Bim. Conversely, knock down of Bim, but not Bcl-2 expression, decreased the susceptibility of tumor cells to paclitaxel-mediated killing. Similar observations were made using a panel of breast and prostate cancer cell lines. Paclitaxel impairs microtubule function, causes G2/M cell cycle blockade, mitochondria damage, and p53-independent apoptosis. These results established Bim as a critical molecular link between the microtubule poison, paclitaxel, and apoptosis.

Manipulating the host response to autologous tumour vaccines.

From our way of thinking the problem facing vaccine strategies for cancer is not that we do not have "enough" tumour antigens. The problem is we cannot induce an immune response that is sufficient to mediate tumour regression. The normal "checks and balances" found in the body prevent the sustained expansion and subsequent persistence of immune killer cells. If vaccine strategies are going to become effective treatments for cancer patients, they will need to overcome this substantial roadblock. Recent developments in immunology have provided insights into the mechanisms that regulate the expansion and persistence of T cells. This has allowed investigators to reinterpret decades-old observations suggesting that chemotherapy administered before vaccination often led to a stronger immune response. This manuscript will review experiments that offer an explanation for these observations and present pre-clinical data from our laboratory that describes an innovative new approach to combining chemotherapy and vaccination. This approach is readily translatable to the clinic and is broadly applicable to any vaccine strategy for advanced cancer.

Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.

Coreceptor utilization by human immunodeficiency virus type 1 is not a primary determinant of neutralization sensitivity.

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses.

Mycosis fungoides and Sezary syndrome are not associated with HTLV-I infection: an international study.

Association between mycosis fungoides (MF), its leukaemic variant Sezary syndrome (SS) and the human T-cell lymphotropic virus type-I (HTLV-I) has been controversial, with the reported incidence of infection varying between 0% and nearly 100%. We studied 127 patients (85 MF, 28 SS, five Sezary cell leukaemia, four lymphomatoid papulosis, and five unspecified cutaneous T-cell lymphomas (CTCL)) originating from Europe (France, Spain, U.K., Portugal) or from U.S.A. (California) for the presence of HTLV-I infection markers. HTLV-I and -II serology were performed on 78 patients using standard immunological methods. Reverse transcriptase (RT) assay was also performed in 26 cases using an RT-PCR-based method of high sensitivity. Molecular analyses were performed on 215 DNA samples (121 from fresh PBMCs, 26 from PBMCs after short-term culture and 68 from skin lesions) by PCR amplification using HTLV-I and -II gag, pol, env, pX and LTR specific primers. Immunological tests were negative except for two sera which were indeterminate. PCR with all HTLV-I and -II primer pairs showed negative results in all 215 samples investigated. No RT activity was detected in short-term PBMC cultures of any of the 26 cases studied. The results of this large study from five different countries clearly indicate that MF and SS are not associated with HTLV-I infection.

Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma.

Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis.

Human T-cell lymphotropic virus type I-associated retinal lymphoma. A clinicopathologic report.

Human T-cell lymphotropic virus type I has been associated with a wide range of ocular conditions, including neoplastic, infectious, and inflammatory lesions. We studied a patient infected with human T-cell lymphotrophic virus type I who presented with deep retinal and subretinal infiltrates but without cells in the vitreous. The differential diagnosis included intraocular lymphoma and fungus infection. A chorioretinal biopsy specimen obtained for tissue diagnosis disclosed large atypical mononuclear cells located primarily at the level of the retinal pigment epithelium but focally involving overlying retina. Electron microscopy of this infiltrate showed features consistent with adult T-cell lymphoma/leukemia. Infection by human T-cell lymphotropic virus type I was verified by polymerase chain reaction studies conducted on peripheral-blood mononuclear cells. This case emphasizes the occurrence of intraocular lesions in adult T-cell lymphoma/leukemia that clinically show some features similar to those of the usual ocular lymphoma (reticulum cell sarcoma); diagnosis can be established by chorioretinal biopsy, thereby allowing appropriate therapy.

IL-10 inhibits HIV-1 replication and is induced by tat.

Interleukin 10 (IL-10) is produced by TH2 lymphocytes and regulates both lymphoid and myeloid cells. In the present study we demonstrate that IL-10 is expressed and produced spontaneously in the peripheral blood mononuclear cells (PBMCs) of all HIV-1 infected individuals tested, 3 of 19 cases of HIV-negative lymphoma and none of five healthy controls. IL-10 mRNA was detectable in both monocytes/macrophages and T lymphocytes isolated from PBMCs of HIV infected patients. We have also shown that infection of promonocytic (U937) and T (H9) cell lines with HIV stimulates IL-10 secretion. Furthermore, a T cell line (H9) stably transfected with a HIV tat expression-vector secreted higher levels of IL-10. We have also demonstrated that rhIL-10 inhibited HIV-1 replication in infected monocytes and PBMCs in a dose dependent manner. IL-10 may thus participate in long latency between HIV-1 infection and development of AIDS.

All-trans retinoic acid for the treatment of AIDS-related Kaposi's sarcoma: results of a pilot phase II study.

Retinoids have anti-tumor activity in several malignant and premalignant conditions. Since Kaposi's sarcoma is regulated by steroid hormones both in vivo and in vitro, we hypothesized that retinoids may have anti-tumor effects in AIDS-related Kaposi's sarcoma. Thus, 27 patients with mucocutaneous, non-visceral AIDS-related Kaposi's sarcoma were treated with all-trans retinoic acid (tRA). Poor tolerance was observed at the initial starting dose of 150 mg/m2, and thus subsequent patients were treated using a weekly dose escalation, starting with 45 mg/m2 (given daily, in subdivided doses), to the target dose of 150 mg/m2 (given daily in three subdivided doses). Nearly half (46%) of the patients had extensive mucocutaneous disease with over 25 lesions. No patient had received prior cytotoxic chemotherapy. Ten patients had CD4 lymphocytes of 200/mm3 or greater (strata I); and 17 had under 200/mm3 CD4 lymphocytes (strata II). The median of the average daily tRA dose administered was 150 mg (90 mg/m2; there was no significant difference in the dose tolerance between the two strata). Adverse effects consisted of transient mild to moderate headaches in 65% of patients, mild to moderate skin dryness and cheilitis in 61%, and nausea and vomiting in 31%. Hematologic toxicities included hypertriglyceridemia in 62%, anemia in 23%, and neutropenia in 23%. Partial response to therapy was observed in 4/24 (17%) evaluable patients, occurring after 12, 20, 24, and 28 weeks of therapy, and lasting 4-24 weeks. Three responders had baseline CD4 lymphocyte counts < 200/mm3. Three additional patients experienced reduction in measured indicator lesions of greater than 25% but less than 50%, and seven patients experienced disease stabilization of 16 weeks or greater. In evaluable patients, the median time to disease progression was 22 weeks and the overall median survival in all patients was 27.3 months. No significant changes in CD4 lymphocyte counts, p24 antigen, and beta 2 microglobulin were observed over time. However, a statistically significant increase was observed in soluble IL-2 receptor levels while on tRA (p = 0.037). We conclude that tRA has activity in patients with mucocutaneous AIDS-related Kaposi's sarcoma with acceptable toxicity. tRA has immunological effects without upregulation of HIV parameters. Additional studies in combinations or with more active retinoids are warranted.

Infectious decay of human immunodeficiency virus type 1 in plasma.

AIDS is caused by the human immunodeficiency virus type 1 (HIV-1). Recent methods have been developed to estimate infectious titer in various bodily fluids, including blood. However, lack of information about HIV-1 stability in blood has restricted the use of these techniques to fresh samples in immediately accessible virology laboratories. In studies of infectious virus decay, it was found that at room temperature, complete decay of infectious HIV-1 in plasma can require > 7 days. Furthermore, the stability of HIV-1 was enhanced by storage at 4 degrees C, suggesting that fresh plasma could be sent on ice to core laboratories for viral quantitation. These studies also emphasize the need for thorough decontamination of all potentially infectious material.

Rapid generation of sequence variation during primary HIV-1 infection.

OBJECTIVE: HIV-1 undergoes extensive genetic variation in infected individuals. The extent of genetic variation has been examined in patients with AIDS, but little is known regarding the appearance of HIV-1 genetic variation immediately following infection during the primary phase of HIV-1 infection prior to seroconversion. DESIGN: We examined HIV-1 genetic variation during this early phase of HIV-1 infection by polymerase chain reaction (PCR) and nucleotide sequence analysis of the V4 by polymerase chain reaction (PCR) and nucleotide sequence analysis of the V4 variable region and the CD4-binding domain. RESULTS: Our results demonstrate that extensive sequence variation is seen early after infection, although a predominant HIV-1 species is maintained. CONCLUSIONS: The type of variants that occur are dynamic, changing over time, and the mutations seen are consistent with those expected from random occurrence, unlike the pattern of variation previously reported during later stages of disease.

Transient high levels of viremia in patients with primary human immunodeficiency virus type 1 infection.

BACKGROUND: The rapidly evolving clinical picture of primary infection with the human immunodeficiency virus type 1 (HIV-1) suggests that a better understanding of the kinetics of viral replication in vivo during the short period before seroconversion may provide insight into the pathogenesis of the acquired immunodeficiency syndrome (AIDS). METHODS AND RESULTS: Titers of infectious HIV-1 were determined by end-point-dilution culture in sequential samples of plasma and peripheral-blood mononuclear cells from four patients with primary infection, with peak titers of 1000 to 10,000 tissue-culture-infective doses per milliliter of plasma and 100 to 10,000 infective doses per 10(6) peripheral-blood mononuclear cells. The high viral burden in mononuclear cells was confirmed by quantitative studies using a polymerase-chain-reaction method. In as little as 10 days, the high HIV-1 load in both plasma and cells decreased spontaneously and precipitously, at least 100-fold, in all four patients. CONCLUSIONS: Although p24 core antigenemia and viral isolation have previously been described during primary HIV-1 infection, this report documents the large viral burden during the acute phase of infection. The rapid and spontaneous decline in the viral load suggests an effective immune response in the host that, if understood, may be used to combat AIDS.

Conformational epitope on gp120 important in CD4 binding and human immunodeficiency virus type 1 neutralization identified by a human monoclonal antibody.

A human monoclonal antibody designated 15e is reactive with the envelope glycoprotein (gp120) of multiple isolates of human immunodeficiency virus type 1 (HIV-1). Antibody 15e also neutralizes HIV-1 with broad specificity and blocks gp120 binding to CD4. Characterization of the 15e epitope shows that it is conformation dependent and is distinct from previously recognized functional domains of gp120, suggesting that this epitope represents a novel site important for HIV-1 neutralization and CD4 binding. These findings have implications for the development of a vaccine for AIDS.

Seroprevalence of human T cell leukemia viruses in selected populations of homosexual men.

This study sought to define the seroprevalence of human T cell leukemia virus (HTLV) types I and II in selected populations of homosexual men. Serum specimens were screened for antibodies to HTLV and to human immunodeficiency virus (HIV) by enzyme immunoassay; successive testing of specimens with positive results was done by Western blotting and radioimmunoprecipitation assay (RIPA) and then by polymerase chain reaction (PCR) assay on available peripheral blood mononuclear cells (PBMC). Of 1290 specimens, only 4 had antibodies against HTLV confirmed by RIPA. PCR analysis of DNA from PBMC from two subjects showed one to be HTLV-I and the other to be HTLV-II; both men also had HIV antibodies. These results demonstrate a lower seroprevalence rate for HTLV than some previous studies and emphasize the need for specific confirmatory tests.

High concentrations of recombinant soluble CD4 are required to neutralize primary human immunodeficiency virus type 1 isolates.

There is substantial evidence supporting the CD4 molecule as the principal cellular receptor for the human immunodeficiency virus type 1 (HIV-1). A number of truncated recombinant soluble CD4 (sCD4) molecules have been produced and shown to easily neutralize infection of laboratory strains of HIV-1 in vitro, and clinical trials using these sCD4 preparations have begun in patients with AIDS. Infectious HIV-1 titers in the plasma and peripheral blood mononuclear cells of five patients receiving sCD4 at 30 mg/day were sequentially monitored. No significant decrease in viral titers was found during therapy. Furthermore, plasma samples from eight patients with AIDS were titrated for HIV-1 with and without the addition of sCD4 ex vivo. Despite the addition of sCD4 at up to 1 mg/ml, there was little change in plasma viral titers. Subsequently, 10 primary HIV-1 isolates were tested for their susceptibility to neutralization in vitro by one preparation of sCD4. Neutralization of these clinical isolates required 200-2700 times more sCD4 than was needed to inhibit laboratory strains of HIV-1. Similar results were observed using one other monomeric sCD4 preparation and two multimeric CD4-immunoglobulin hybrid molecules. We conclude that unlike laboratory strains, primary HIV-1 isolates require high concentrations of sCD4 for neutralization. This phenomenon may pose a formidable problem for sCD4-based therapeutics in the treatment of HIV-1 infection.

CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.

CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1.

One neuronal cell line (SK-N-MC) was found to be susceptible to productive infection by multiple isolates of the human immunodeficiency virus type 1 (HIV-1). Characterization of SK-N-MC cells showed that these cells are neuroectodermal in origin in that they express dopamine hydroxylase, catecholamines, neuron-specific enolase, and neurofilaments. Despite their susceptibility to HIV-1 infection, SK-N-MC cells had no detectable CD4 and this infection was not blocked by anti-CD4 monoclonal antibodies (OKT4A, Leu3A) or recombinant soluble CD4. These experiments demonstrated that certain cells of neuroectodermal origin are susceptible to infection in vitro by HIV-1 via a CD4-independent mechanism.

Quantitation of human immunodeficiency virus type 1 in the blood of infected persons.

We used end-point-dilution cultures to measure the level of infectious human immunodeficiency virus type 1 (HIV-1) in peripheral-blood mononuclear cells (PBMC) and plasma of 54 infected patients who were not receiving antiviral chemotherapy. HIV-1 was recovered from the plasma and PBMC of every seropositive patient, but from none of 22 seronegative control subjects. The mean titers in plasma were 30, 3500, and 3200 tissue-culture-infective doses (TCID) per milliliter for patients with asymptomatic infection, the acquired immunodeficiency syndrome (AIDS), and the AIDS-related complex, respectively. In PBMC, the mean titers were significantly higher for symptomatic patients (AIDS, 2200, and AIDS-related complex, 2700 TCID per 10(6) PBMC) than asymptomatic patients (20 TCID per 10(6) PBMC). The values for the symptomatic patients were considered to indicate that at least 1 in 400 circulating mononuclear cells harbored HIV-1. The HIV-1 titers of seven patients with AIDS or AIDS-related complex treated with zidovudine for four weeks decreased significantly in plasma but not in PBMC. In addition, the mean titer in the plasma of 20 patients receiving long-term zidovudine treatment (130 TCID per milliliter) was 25-fold lower than the mean for comparable untreated patients with AIDS or AIDS-related complex. We conclude that the levels of HIV-1 in plasma and PBMC are much higher than previous estimates. This high degree of HIV-1 viremia raises the possibility that the direct cytopathic effect of this retrovirus alone may be sufficient to explain much of the pathogenesis of AIDS.