Rapid detection of engraftment using T cell receptor gene polymorphism after allogeneic bone marrow transplantation in an alloimmunized child with severe aplastic anemia

A severely alloimmunized boy with aplastic anemia received an HLA-identical BMT from his brother. Despite intensive immunosuppression and large marrow dose, peripheral signs of engraftment occurred only late under G-CSF treatment. With leukocyte counts of < 0.5 x 10(9)/l, chimerism could be proven not only by oligonucleotide fingerprinting but also within 48 h by analysis of polymorphism in the TCR gene family. This rapid and sensitive method to detect engraftment before it became quantitatively evident was important for the clinical management of the patient, obviating the need to search for an alternative marrow donor.     

T cell receptor V-segment frequencies in aged individuals

The T V alpha and V beta cell specificities repertoire usage in aging subjects was studied by the use of seven different monoclonal antibodies specific for defined regions of the T cell receptor (TCR). Except for the V beta 8 subfamily, no differences were observed in the frequency of T cells bearing selected V alpha beta epitopes, between old and control subjects.     

Transitional cell carcinoma of the bladder in an HIV-infected patient

A case of transitional cell carcinoma of the urinary bladder in a patient infected with the human immunodeficiency virus is reported. The tumour was very large, occupying most of the bladder, however cystoscopic removal of the tumour was successful with no recurrence after one year. This association has never reported before. The possible contribution of human immunodeficiency virus infection to its occurrence is discussed.     

Therapeutic levels of human protein C in rats after retroviral vector-mediated hepatic gene therapy

Protein C deficiency results in a thrombotic disorder that might be treated by expressing a normal human protein C (hPC) gene in patients. An amphotropic retroviral vector with a liver-specific promoter and the hPC cDNA was delivered to rat hepatocytes in vivo during liver regeneration. Expression of hPC varied from 55 to 203 ng/ml (1.3-5.0% of normal) for 2 wk after transduction. Expression increased to an average of 900 ng/ml (22% of normal) in some rats and was maintained at stable levels for 1 yr. All of these rats developed anti-hPC antibodies and exhibited a prolonged hPC half-life in vivo. The hPC was functional as determined by a chromogenic substrate assay after immunoprecipitation. We conclude that most rats achieved hPC levels that would prevent purpura fulminans, and that hepatic gene therapy might become a viable treatment for patients with severe homozygous hPC deficiency. Anti-hPC antibodies increased the hPC half-life and plasma levels in some rats, but did not interfere with its functional activity. Thus, the development of antibodies against a plasma protein does not necessarily abrogate its biological effect in gene therapy experiments.     

Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene

Replication-deficient adenovirus (Ad) vectors are effective to specifically target the respiratory epithelium for either corrective gene therapy such as cystic fibrosis or for mucosal immunization. As a consequence of transducing the lower respiratory tract with an E1/E3 deleted Ad5 vector, host responses have been characterized by the duration of transgene expression and by the induction of CTL responses. However, limited emphasis has been devoted to understanding the contribution of CD4+ T cell responses to the Ad vector. Both CD4+ and CD8+ T cells migrate into the lung following sequential intratracheal Ad5 transgene instillations. Isolated CD3+ T lymphocytes from the lungs were predominantly of the Th2 type, and after cell sorting, the IL-4-producing T cells were largely CD4+, while IFN-gamma expression was associated with both CD4+ and CD8+ T cells. Ab responses to the Ad5 vector and to the expressed transgene beta-galactosidase (beta gal) revealed elevated bronchial and serum IgA and IgG Abs with low neutralization titers. Analysis of serum IgG subclass responses showed IgG1 and IgG2b with lower IgG2a Abs to Ad5 and IgG2a and IgG2b Ab responses to beta gal. Ad5-specifc CD4+ T cells produced both Th1 (IFN-gamma and IL-2)- and Th2 (IL-4, IL-5, IL-6)-type cytokines, while beta gal-specific CD4+ T cells secreted IFN-gamma and IL-6. This study provides direct evidence for the concomitant induction of Th2- with Th1-type responses in both the pulmonary systemic and mucosal immune compartments to the Ad5 vector as well as a Th1-dominant response to the transgene.     

Alpha beta T cell response to Toxoplasma gondii in previously unexposed individuals

The mechanisms by which T cells from previously unexposed hosts respond in vitro to certain intracellular pathogens remain to be fully understood. We report and characterize the in vitro reactivity to Toxoplasma gondii of human alpha beta T cells from T. gondii-seronegative individuals. Resting alpha beta T cells from these individuals proliferated in response to PBMC infected with T. gondii or pulsed with T. gondii lysate Ags. This was accompanied by an increase in the percentage of CD4+ alpha beta T cells. Purified CD4+ alpha beta T cells but not CD8+ alpha beta T cells proliferated in response to these T. gondii preparations. Both CD4+ alpha beta T cells with naive (CD45RA+) and memory (CD45RO+) phenotypes from adults as well as alpha beta T cells from T. gondii-seronegative newborns proliferated after incubation with T. gondii. This alpha beta T cell response to the parasite was inhibited by anti-HLA-DR mAb and to a lesser degree by anti-HLA-DQ mAb. Use of paraformaldehyde-fixed PBMC completely abrogated the proliferation of alpha beta T cells, indicating the need for processing of T. gondii Ags. Analysis of the TCR V beta expression did not show evidence for restriction in TCR V beta usage during T. gondii stimulation of alpha beta T cells. Alpha beta T cells secreted significant amounts of IFN-gamma after incubation with T. gondii-infected monocytes. This rapid and remarkable alpha beta T cell response may play an important role in the early events of the immune response to T. gondii.     

Treatment and prophylaxis of disseminated Mycobacterium avium complex in HIV-infected individuals

OBJECTIVE: To review the pathophysiology, epidemiology, treatment, and prophylaxis of disseminated Mycobacterium avium complex (MAC) infection in HIV-infected individuals. DATA SOURCES: A MEDLINE (January 1966-July 1997) and AIDSLINE (January 1980-July 1997) search of basic science articles pertinent to the MAC infection in HIV-infected patients. STUDY SELECTION AND DATA EXTRACTION: All articles were considered for possible inclusion in the review. Pertinent information, as judged by the authors, was selected for discussion. DATA SYNTHESIS: The organism, epidemiology, and pathophysiology of disseminated MAC are discussed for background. A review of clinical trials for the treatment and prophylaxis of disseminated MAC are presented, along with unresolved issues concerning these topics. CONCLUSIONS: The incidence of disseminated MAC has increased dramatically with the AIDS epidemic. The infection can lead to increased morbidity and mortality in HIV-infected patients. Treatment regimens for patients with a positive culture for MAC from a sterile site should include two or more drugs, including clarithromycin. Prophylaxis against disseminated MAC should be considered for patients with a CD4 cell count of less than 50/mm3.     

A retroviral vector that directs simultaneous expression of alpha and beta T cell receptor genes

The transfer of alpha/beta T cell receptor (TCR) genes into T lymphocytes or their precursors could provide a means to increase frequency of tumor- or pathogen-specific cytotoxic T lymphocytes. To begin to address this possibility, we have used class I MHC-restricted alpha/beta TCR cDNAs to develop a retroviral TCR expression vector. Alpha- and beta-chain cDNAs were inserted into a derivative of the LN series of retroviral vectors, with the retroviral LTR directing expression of TCR-beta and an internal cytomegalovirus promoter/enhancer driving TCR-alpha expression. The variable region fragments can be replaced using unique restriction sites that have been introduced into the proximal constant regions. We have used this vector system to transfer two different pairs of alpha/beta TCR genes into an alpha- and beta-chain-deficient T cell hybridoma. TCR- hybridoma cells were transduced by coculture with pools of virus-producing cells, and fluorescence-activated cell sorting was used to enrich for cells expressing the transduced TCR. Transduction with either alpha/beta TCR restores stable, long-lived expression of the alpha/beta TCR complex. TCR-mediated signal transduction is also reconstituted, as demonstrated by the ability of transduced cells to secrete IL-2 following stimulation with Vbeta-specific antibodies. Our results suggest that alpha/beta T cell receptor gene transfer could provide a basis for new approaches to immunotherapy, and that further studies examining the in vivo fate of transduced TCR are possible.     

Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations

The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of HIV-1-specific CTLs in the semen of HIV-infected individuals

CTLs play an important role in controlling cell-associated HIV. Since the majority of HIV infections are acquired through sexual transmission, we investigated whether antiviral CTLs were present in the male urogenital tract using semen as a source of T cells. We were able to establish anti-HIV cytolytic lines in five of five HIV-infected men with CD4 counts of >500/microl, although cloning efficiencies were lower than with peripheral blood-derived T cells. CTLs generated from the semen of three men were analyzed in detail and showed a broadly active response, recognizing gag, env, and pol proteins. Detailed analysis of two gag-specific clones from one of the individuals demonstrated HLA class I restriction and recognition of the same p24 epitope (EQASQEVKNWMT). In summary, our results demonstrate the presence of a broad CTL response to HIV in the urogenital tract and provide a rationale for further studies of local enhancement of genital mucosal responses by anti-HIV immunization.     

T cell receptor gene deletion circles identify recent thymic emigrants in the peripheral T cell pool

Progenitor cells undergo T cell receptor (TCR) gene rearrangements during their intrathymic differentiation to become T cells. Rearrangements of the variable (V), diversity (D), and joining (J) segments of the TCR genes result in deletion of the intervening chromosomal DNA and the formation of circular episomes as a byproduct. Detection of these extrachromosomal excision circles in T cells located in the peripheral lymphoid tissues has been viewed as evidence for the existence of extrathymic T cell generation. Because all of the T cells in chickens apparently are generated in the thymus, we have employed this avian model to determine the fate of the V(D)J deletion circles. In normal animals we identified TCR Vgamma-Jgamma and Vbeta-Dbeta deletion circles in the blood, spleen, and intestines, as well as in the thymus. Thymectomy resulted in the gradual loss of these DNA deletion circles in all of the peripheral lymphoid tissues. A quantitative PCR analysis of Vgamma1-Jgamma1 and Vbeta1-Dbeta deletion circles in splenic gamma delta and Vbeta1(+) alphabeta T cells indicated that their numbers progressively decline after thymectomy with a half-life of approximately 2 weeks. Although TCR deletion circles therefore cannot be regarded as reliable indicators of in situ V(D)J rearrangement, measuring their levels in peripheral T cell samples can provide a valuable index of newly generated T cells entering the T cell pool.     

The prevalence of oral candidiasis in HIV-infected individuals and dental attenders in Edinburgh

This study prospectively assessed the prevalence of oral candidal carriage and oral candidiasis in known HIV-seropositive individuals (n = 121) and other dental attenders in Edinburgh (n = 614). Candida species were isolated from 57.4% of dental attenders and 93.4% of HIV-seropositive subjects. Clinical evidence of oral candidiasis was observed in 6% and 52% of these groups respectively, erythematous forms of candidiasis being the commonest in both groups.     

Studies of T cell reconstitution after hematopoietic stem cell transplant

Hematopoietic stem cell (HSC) transplantation, whatever its conditions, is associated with an increased risk of infections and tumoral complications, because of a delayed immune reconstitution. T-cell regeneration has been mostly investigated and appears to come more from graft and/or host mature T-cells, rather than from the differentiation/maturation of reinfused progenitors. In allogeneic setting, the immune defect is enhanced by the immune host/donor conflict and the use of prophylactic or curative immunosuppressive therapy. The tools used for studying post-transplant immunity are the following: immunophenotyping (kinetics and alterations of lymphocyte subset reconstitution), functional studies of T cell proliferation, cytokine production, cytotoxicity and signal transduction, as well as studies of T cell repertoire diversity. The CD4/CD8 cell immunophenotyping might be enough for routine clinical evaluation, allowing an adapted prophylaxis of opportunistic infections in those immune-suppressed patients, while functional assays might be useful to evaluate the persistence overtime of defects in immune reconstitution. These overall assays are useful both for basic and clinical research and allow better understanding in the mechanisms for T cell regeneration in the diverse types of HSC transplants performed nowadays particularly after graft of purified HSC where immune reconstitution remains a key question.     

Short-term stability of HIV provirus levels in the peripheral blood of HIV-infected individuals

Changes in viral load have been reported to reflect disease progression or response to therapy; however, the stability of HIV DNA levels in HIV-infected individuals has not been extensively studied. Cellular HIV DNA levels in infected individuals were evaluated over a short time period to determine degree of variability as well as any correlation with other measurements of virus load or immune status. Peripheral blood mononuclear cells (PBMC) were obtained several times over 1 month from 32 asymptomatic or symptomatic non-AIDS, HIV-infected individuals currently on AZT therapy. PCR amplification of the HIV gag region was performed with DNA from PBMC lysates and the PCR amplified products quantitated by liquid phase hybridization. HIV DNA levels in the majority of the patients were relatively stable, with 26 of 32 persons having less than threefold change. Changes over the study period were both positive and negative, and the median change in HIV DNA levels was 68.6%. These changes were found to positively correlate with fluctuations in plasma p24 levels. In contrast, no correlations were found with other measurements of immune system activity, including changes in CD4 number, CD4 percent, and beta 2-microglobulin when compared with provirus changes. This study shows that levels of HIV DNA can be relatively stable over short periods in most non-AIDS, HIV-infected persons.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cells transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho+/++ (dull/bright) and Rho- (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho+/++ cell population contained > 99% of committed progenitor cells with in vitro colony-forming ability. The Rho- cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho- stem cells or purified Rho- stem cells in combination with large numbers of Rho+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho- stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Interferon-alpha gene therapy for cancer: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells are both effective strategies for gene delivery in murine tumor models

Stable transfection of tumor cells with IFN-alpha genes has been shown to result in abrogation of tumor establishment and induction of antitumor immunity. However, strategies suitable for the clinical application of IFN-alpha gene therapy for cancer have not been reported. In this study, we investigated two gene delivery systems capable of mediating the local paracrine production of high levels of biologically active IFN-alpha in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-alpha, it was possible to obtain stable retroviral producer cell lines and transduce a variety of murine tumor cells including syngeneic fibroblasts to stably secrete 2000-5000 U (40-100 ng) murine IFN-alpha/10(6) cells/24 h. IFN-alpha transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-alpha gene delivery using retrovirally transduced syngeneic fibroblasts was capable of suppressing the establishment of the poorly immunogenic TS/A mouse mammary adenocarcinoma and induced antitumor immunity. Particle mediated transient transfection of tumor cells using the gene gun led to the production of up to 20,000 U IFN-alpha/10(6) cells during the first 24 h and proved to be equally effective in suppressing establishment of TS/A adenocarcinoma and inducing antitumor immunity. These results suggest that retroviral transduction of autologous fibroblasts can serve as an effective gene delivery method for IFN-alpha gene therapy of cancer. Particle-mediated transfection of freshly isolated tumor cells may represent a clinically attractive alternative approach for nonviral gene delivery. Both strategies circumvent the difficulties in routinely establishing primary tumor cell lines from the vast majority of human cancers.     

Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line

Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.     

T cell engraftment in lymphoid tissues of human peripheral blood lymphocyte reconstituted SCID mice with or without prior activation of cells

A controlled study was undertaken to determine the stability of LSD in pooled urine samples. The concentrations of LSD in urine samples were followed over time at various temperatures, in different types of storage containers, at various exposures to different wavelengths of light, and at varying pH values. LSD concentrations were measured quantitatively by the Abuscreen RIA and by HPLC using a fluorescence detection method. Good correlation was observed between the immunoassay and the fluorescent integrity of the LSD molecule. Thermostability studies were conducted in the dark with various containers. These studies demonstrated no significant loss in LSD concentration at 25 degrees C for up to 4 weeks. After 4 weeks of incubation, a 30% loss in LSD concentration at 37 degrees C and up to a 40% at 45 degrees C were observed. Urine fortified with LSD and stored in amber glass or nontransparent polyethylene containers showed no change in concentration under any light conditions. Stability of LSD in transparent containers under light was dependent on the distance between the light source and the samples, the wavelength of light, exposure time, and the intensity of light. After prolonged exposure to heat in alkaline pH conditions, 10 to 15% of the parent LSD epimerized to iso-LSD. Under acidic conditions, less than 5% of the LSD was converted to iso-LSD. We also demonstrated that trace amounts of metal ions in buffer or urine could catalyze the decomposition of LSD and that this process can be avoided by the addition of EDTA. This study demonstrates the importance of proper storage conditions of LSD in urine in order to insure proper analytical testing results over time.     

Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline

Evolution of HIV-1 env sequences was studied in 15 seroconverting injection drug users selected for differences in the extent of CD4 T cell decline. The rates of increase of either sequence diversity at a given visit or divergence from the first seropositive visit were both higher in progressors than in nonprogressors. Viral evolution in individuals with rapid or moderate disease progression showed selection favoring nonsynonymous mutations, while nonprogressors with low viral loads selected against the nonsynonymous mutations that might have resulted in viruses with higher levels of replication. For 10 of the 15 subjects no single variant predominated over time. Evolution away from a dominant variant was followed frequently at a later time point by return to dominance of strains closely related to that variant. The observed evolutionary pattern is consistent with either selection against only the predominant virus or independent evolution occurring in different environments within the host. Differences in the level to which CD4 T cells fall in a given time period reflect not only quantitative differences in accumulation of mutations, but differences in the types of mutations that provide the best adaptation to the host environment.