Allogeneic haematopoietic stem cell transplantation in patients with human immunodeficiency virus: the experiences of more than 25 years.

For treatment of several malignancies, transplantation of allogeneic haematopoietic stem cells (HSCT) derived from bone marrow or peripheral blood has been used as a therapeutic procedure for decades. In the past, HSCT has been suggested as a treatment option for infection with the human immunodeficiency virus type 1 (HIV-1), but these attempts were mostly unsuccessful. Today, after the introduction of an active anti-retroviral therapy, the lifetime expectancy of HIV-infected patients has improved substantially, but nevertheless the incidence rate of malignancies in these patients has increased considerably. Therefore, it can be assumed that there will be a rising necessity for HIV-1-infected patients with malignancies for allogeneic HSCT. At the same time, there is increasing interest in treatment methods which might target the HIV-1 reservoir more effectively, and the question has been raised as to whether allogeneic HSCT could be linked to such strategies. In this paper the data of more than 25 years experience with allogeneic HSCT in patients with HIV-1 are reviewed and analysed.

Ribozymes as potential anti-HIV-1 therapeutic agents.

Certain RNA molecules, called ribozymes, possess enzymatic, self-cleaving activity. The cleavage reaction is catalytic and no energy source is required. Ribozymes of the "hammerhead" motif were identified in plant RNA pathogens. These ribozymes possess unique secondary (and possibly tertiary) structures critical for their cleavage ability. The present study shows precise cleavage of human immunodeficiency virus type 1 (HIV-1) sequences in a cell-free system by hammerhead ribozymes. In addition to the cell-free studies, human cells stably expressing a hammerhead ribozyme targeted to HIV-1 gag transcripts have been constructed. When these cells were challenged with HIV-1, a substantial reduction in the level of HIV-1 gag RNA relative to that in nonribozyme-expressing cells, was observed. The reduction in gag RNA was reflected in a reduction in antigen p24 levels. These results suggest the feasibility of developing ribozymes as therapeutic agents against human pathogens such as HIV-1.

Bone marrow transplantation for hematologic malignancies in patients aged 30 years or older.

During the past 10 years, 86 patients 30 to 54 years of age with hematologic malignancies were prepared with high-dose radiochemotherapy and received histocompatible bone marrow grafts. Thirty-four of these patients are surviving for 4 months to 9 years (median, 26 months) following marrow transplantation and 32 of them are in continuing complete remission (CR). Disease-free survival is 44% for 37 patients who were in first remission of acute leukemia or in the chronic phase of chronic granulocytic leukemia (CGL), 23% for 39 patients whose leukemia had relapsed at least once before transplantation or who had advanced stages of CGL, and 60% for ten patients who had hematologic malignancies other than leukemia. The median age of the surviving 34 patients is 36 years (range, 30 to 43 years). The incidence of moderate to severe acute graft-v-host disease (GVHD) was 48% and of chronic GVHD, 26%. The major causes of failure were interstitial pneumonia in 31 patients (24 of whom had antecedent acute GVHD) and recurrent leukemia in 12 patients (11 of whom had either never entered a CR or had relapsed at least once with acute leukemia or had progressive CGL before transplantation). Our data warrant further prospective studies in patients with hematologic malignancies who are older than 30 years.

Comparison of amphotropic and pseudotyped VSV-G retroviral transduction in human CD34+ peripheral blood progenitor cells from adult donors with HIV-1 infection or cancer.

In this study we compared the transduction efficiency of conventional amphotropic MoMLV (LPONL[A]) with the MoMLV pseudotyped with that of VSV-G (LPONL[G]) in peripheral blood progenitor cells (PBPCs) from cancer patients and human immunodeficiency virus (HIV)-infected donors. The results showed that LPONL(A) and LPONL(G) infected the progenitor cells from these sources with equal efficiencies. The transgene neoR was detectable by polymerase chain reaction assay in colonies from 14-day colony-forming unit (CFU) assays and in those derived from long-term culture-initiating cell (LTC-ICs) assays. Although the overall levels of transduction efficiency were similar in cord blood and PBPCs from noninfected cancer donors (25-22%) when either LPONL(G) or LPONL(A) was used, they were significantly lower in HIV-1-infected donors compared with noninfected cancer donors when LPONL(G) was used (13 vs. 25%; p = 0.027), and when LPONL(A) was used (12 vs. 22%; p = 0.087). The clonogenic potentials of infected and noninfected CD34+ cells were similar; thus no toxicity could be attributed to the virus preparation. We conclude that PBPCs from HIV-1-infected individuals are transduced less efficiently than those from non-HIV-infected cancer donors. Nonetheless, PBPCs from HIV-infected persons serve as potential targets in gene therapy for acquired immune deficiency syndrome.

Epidemiology and pathogenesis of cytomegalovirus disease.

Two distinct processes contribute to the spectrum of human cytomegalovirus (HCMV)-induced pathology. In the first instance, cytopathic effects appear to occur as a direct result of virus replication. This type of disease is characterized by persistent HCMV infection of neural or gastrointestinal tissue, which results in HCMV retinitis, encephalitis, hepatitis, or gastroenteritis. Direct cytopathic effects of HCMV are associated with congenitally acquired or acquired immune deficiency syndrome-related manifestations of HCMV infection. A second type of HCMV-associated disease process is driven by immunopathologic mechanisms and results in variable mononucleosis-like syndromes and/or pneumonia in normal or partially immunosuppressed individuals. Human cytomegalovirus-associated interstitial pneumonia appears to derive from a combination of these two types of disease processes. Here, persistent viral infection, immunopathologic mechanisms, and virus-induced expression or repression of cellular genes each constitutes an important factor in pathogenesis. An understanding of the multiple underlying mechanisms of pathogenesis is crucial to devising optimum treatment approaches.

Inhibition of HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes.

Gene therapy for AIDS requires the identification of genes which effectively inhibit HIV-1 replication coupled to an efficient vector system for gene delivery and expression. Hammerhead ribozymes are RNA molecules capable of catalytic cleavage of complementary RNA molecules. Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave HIV RNA in the tat gene (TAT) or in a common exon for tat and rev (TR). The ribozymes were cloned into the LN (LTR-neomycin) retroviral vector plasmids and expressed as part of viral LTR-driven transcripts. The vectors were packaged as amphitropic virions and used to transduce human T-lymphocytes. Expression of the vector transcripts containing the ribozyme sequences was readily detected by Northern blot analysis of the transduced T cells. The T-lymphocytes expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication. In contrast, cells expressing mutant ribozymes, containing substitutions of a key nucleotide in the catalytic domain which cripples the cleavage activity of the ribozymes, supported replication of HIV-1, demonstrating that the functional ribozymes were cleaving the target RNAs. These studies demonstrate that retrovirally transduced ribozymes included in long, multifunctional transcripts, can inhibit HIV replication in human T-lymphocytes. The ribozyme and expression strategies described here should be useful for the gene therapy of AIDS by conferring resistance to HIV-1 replication on cells derived from transduced hematopoietic stem cells.

Seroepidemiologic studies of cytomegalovirus and Epstein-Barr virus infections in relation to human immunodeficiency virus type 1 infection in selected recipient populations. Transfusion Safety Study Group.

Antibodies to human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) were evaluated among 1,171 persons with and without antibodies to human immunodeficiency virus type 1 (anti-HIV-1). These included 97 blood donors, 577 persons given blood components or products, and 497 controls. A significantly higher proportion of anti-HIV-1 positive than -negative donors were anti-CMV-positive, a finding associated with homosexual contact among some of the former. Among subjects with treated clotting disorders, there was no difference in prevalence of anti-CMV or anti-EBV between anti-HIV-1-positive and -negative persons. The prevalence of antibodies to EBV early antigens showed no relationship to anti-HIV-1 status. Anti-CMV positivity in anti-HIV-1-negative donors was associated with an increase in mean CD8 counts and lower mean CD4/CD8 ratio. Anti-CMV and anti-EBV positivity in anti-HIV-1-positive subjects with treated clotting disorders was not associated with a lower CD4 or higher CD8 count than HIV-1 infection alone. Subjects who developed AIDS after enrollment had no significant difference in median time from entry to diagnosis when analyzed by serologic evidence of CMV and EBV antibody status at entry, and a few subjects had AIDS at entry without serologic evidence of prior CMV or EBV infection. The overall results are consistent with acquisition and progression of HIV-1 independently of coincident CMV or EBV infection.

Plasma polymerase chain reaction for cytomegalovirus DNA after allogeneic marrow transplantation: comparison with polymerase chain reaction using peripheral blood leukocytes, pp65 antigenemia, and viral culture.

In a prospective longitudinal study, detection of cytomegalovirus (CMV) DNA in plasma (plasma polymerase chain reaction [PCR]) was compared with PCR of CMV DNA in peripheral blood leukocytes (PBL PCR), the CMV pp65 antigenemia assay, and viral cultures from blood, urine, and throat of 29 patients, 14 of whom received pp65 antigenemia-guided early ganciclovir treatment and 15 of whom received ganciclovir at engraftment. Among 328 blood samples tested by all methods, PBL PCR was the most sensitive test, followed by the pp65 antigenemia assay, plasma PCR, and viremia. In the 14 patients who received pp65 antigenemia-guided early treatment, the incidence of PBL PCR, pp65 antigenemia, plasma PCR, and viremia before day 100 was 79%, 79%, 71%, and 27%, respectively, with a median day of onset of day 32, 42, 45, and 51, respectively. Nine patients (64%) became positive by PBL PCR, pp65 antigenemia, and plasma PCR. Of 15 patients who were treated with ganciclovir at engraftment, 12 (80%) became positive by PBL PCR, plasma PCR, and/or pp65 antigenemia while receiving ganciclovir; 3 (20%) had breakthrough infection with all three methods, including 2 with high-grade antigenemia (more than three positive cells in duplicate staining); none of these patients subsequently developed positive CMV cultures or disease. In 49 specimens, PBL PCR and/or pp65 antigenemia assay could not be performed because of insufficient neutrophil counts. In conclusion, the sensitivity of plasma PCR is significantly lower than that of PBL PCR but similar to that of the pp65 antigenemia assay. Plasma PCR may be particularly useful in clinical situations in which a less sensitive and possibly more specific assay is warranted or in which leukocyte counts are inadequate to perform cell-based assays.

Bone marrow transplantation for acute nonlymphoblastic leukemia during first complete remission. An analysis of prognostic factors.

Sixty-nine patients with acute nonlymphocytic leukemia in first remission received total-body irradiation and chemotherapy followed by allogeneic bone marrow transplantation from histocompatible sibling donors. Patient age was between 1 and 41 years: 20 patients 1-19 years (group 1); 27 patients 20-29 years (group 2); and 22 patients 30-41 years (group 3). Two pretransplant radiochemotherapy regimens were employed: The first 45 patients received total-body irradiation (in a single dose) with cytosine arabinoside and cyclophosphamide; the next 24 patients received total-body irradiation (in a fractionated schedule) with cyclophosphamide alone. For all patients, actuarial disease-free survival is 51% (37 of 69 patients are alive and in continuous remission between 5 months and 9.3 years, median 3.7 years). For group 1 actuarial survival is 56%, group 2 48%, and group 3 48%. When analyzed for pretransplant factors that might predict disease-free survival after bone marrow transplantation neither patient age, white cell count at the time of diagnosis, FAB leukemic subtype, length of time before achieving remission, nor length of time between remission and bone marrow transplantation were established as prognostic.

Genomic locus for a 140 kDa structural protein (pp150) of human cytomegalovirus in strains Towne and AD169.

An HCMV specific clone was isolated from a genomic library of human cytomegalovirus (HCMV) DNA cloned into the expression vector lambda gt11. This clone (lambda 111-1) expressed an HCMV/beta-galactosidase fusion protein which was reactive with rabbit antibody prepared against purified HCMV virions and dense bodies as well as human HCMV immune serum. By probing Western blots of HCMV virion proteins or HCMV-infected cells with antibody prepared against the fusion protein, the authentic gene product of clone lambda 111-1 was identified as a high molecular weight polypeptide of 140. Probing the restriction digests of HCMV DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence for the 140 kDa polypeptide to the long unique region (map coordinates of 0.16-0.18) on HCMV Towne and AD169 genomes.

Ribozymes as anti-HIV-1 therapeutic agents: principles, applications, and problems.

An emerging strategy in the treatment of viral infections is the use of antisense DNA or RNA to pair with, and block expression of viral transcripts. RNA, in addition to being an informational molecule, can also possess enzymatic activity. Thus, by combining anti-sense and enzymatic functions into a single transcript, it is now possible to design catalytic RNAs, or ribozymes, which can specifically pair with virtually any viral RNA, and cleave the phosphodiester backbone at a specified location, thereby functionally inactivating the viral RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. There are several different catalytic motifs which possess enzymatic activity, and each one of these can be incorporated into an enzymatic antisense with site-specific cleavage capabilities. By focusing on one type of catalytic motif, the hammerhead, we describe the principles behind the development of ribozymes as transacting, site-specific ribonucleases, several applications of ribozymes in functional destruction of target RNAs, as well as several of the problems confronting their use. We also describe a liposome delivery system which facilitates intracellular inclusion of ribozymes, and may provide a means for therapeutic delivery of ribozymes to HIV-1 infected cells.

Pretransplant pulmonary function predicts cytomegalovirus-associated interstitial pneumonia following bone marrow transplantation.

STUDY OBJECTIVE: To determine the value of pulmonary function tests (PFTs) in predicting the development of human cytomegalovirus (CMV)-associated interstitial pneumonia (IP) in allogeneic bone marrow transplant (BMT) recipients. DESIGN: Nonrandomized, prospective, open-trial study. SETTING: Tertiary referral medical center. PATIENTS: 66 evaluable CMV-seropositive patients with hematologic malignancies who were undergoing allogeneic BMT. INTERVENTION: FEV1, FVC, FEV1/FVC, TLC, Dcoc/VA, PaO2, and P(A-a)O2 were measured on days -13, +33, and +44 following BMT. CMV-IP was diagnosed when typical roentgenographic findings developed with confirmatory positive bronchoalveolar lavage (BAL) using standard cytologic and/or rapid culture techniques. MEASUREMENT AND MAIN RESULTS: Univariate logistic regression analysis to predict the development of CMV-IP revealed significant associations with the day -13 and +33 percent predicted FEV1, FVC, and TLC (p < 0.01) but no associations with other PFT parameters or with changes in these parameters. Stepwise logistic regression analysis demonstrated that only BAL positivity for CMV (odds ratio 14.8; p = 0.0002) and day -13 percent predicted FEV1 (odds ratio 0.92; p = 0.0004) were significant independent predictors of CMV-IP. CONCLUSION: Pretransplant lung function is a previously unrecognized strong predictor and risk factor for the subsequent development of CMV-IP in BMT recipients.

Prevention and management of CMV-related problems after hematopoietic stem cell transplantation.

Prevention and management of human cytomegalovirus (CMV) infection after hematopoietic stem cell transplantation has improved substantially in the past decade. However, with this improvement, there is increased complexity in deciding which diagnostic tests, treatment strategies and immunologic assessments are optimal for different patient populations. The purpose of this review is to address certain practical problems that commonly arise and suggest a suitable approach to management that should have wide applicability.

Confirmation of HIV infection using gene amplification.

Direct recognition of viral gene sequences can be used to detect human immunodeficiency virus (HIV-1) in clinical specimens. A modification of the polymerase chain reaction (PCR) for amplification of gene sequences was used for detection of HIV-1-specific RNA prepared from peripheral blood mononuclear cells (PBMC). The RNA served as a template for reverse transcriptase using primers derived from both the 3'ORF and the LTR regions of HIV-1, as well as from the control cellular sequences encoding beta-actin and T cell receptor. The resultant DNA was amplified with DNA polymerase. A transcriptional step using the bacteriophage T7 promoter recognition sequences, incorporated into the primers, was used to enhance the efficiency of the amplification process. This assay detects as few as 100 RNA copies of cloned HIV-1 genome. Starting with 1 microgram RNA isolated from PBMC, we were able to detect HIV-1 sequences in patients with symptomatic and asymptomatic HIV-1 infection. The inclusion of T cell-specific primers permitted simultaneous evaluation of an immunologic parameter. The PCR can be applied to RNA samples for detection of viral and cellular sequences and is a rapid and efficient means for detection of HIV-1 sequences as well as potentially informative cellular sequences.

Cytomegalovirus infection in the bone marrow transplant recipient.

Over the past 5 years, with the introduction of preventive ganciclovir therapy and a better understanding of the immunology of cytomegalovirus (CMV) in bone marrow transplant (BMT) recipients, there has been a significant change in the management of CMV infection. As the field of BMT has moved into this era of prophylaxis, there are new problems posed by CMV infection that require additional attention. The natural course of CMV-associated disease is undergoing a change, with a frame-shifting of disease onset to later times after BMT. Yet, the success of this antiviral prophylaxis has been of central importance of new developments in marrow transplantation. At the same time that these new antiviral approaches have developed, there has been intensive interest in reducing the cost of BMT. With this, there is a concern for use of the available antiviral strategies in the most efficient manner. This article reviews the various management options relating to control of CMV and discusses certain areas relating to new research strategies that promise to provide even better approaches to the problem of CMV in this population.

A prospective analysis of nosocomial wound infection after mastectomy.

We evaluated the postoperative course of all patients who had mastectomies from 1978 through 1982 at City of Hope National Medical Center (Duarte, Calif). The overall clean mastectomy wound infection rate was 24/294 (8.2%). The incidence of mastectomy wound infection varied with the method of biopsy and was 3.2% after needle aspiration and 9.5% after open biopsy. Mastectomy immediately after open biopsy ("one step") had an infection rate of 5.3%, whereas mastectomy at a subsequent procedure ("two step") had a rate of 12.4%. The maximal infection rate (23.0%) occurred following the two-step procedure when the interval was four to seven days. The infection rates for patients hospitalized three or more days before mastectomy were elevated, but no significant correlation was observed between the infection rate and other demographic factors. We recommend that needle aspiration biopsy be used prior to open biopsy to minimize the need for a two-step approach to mastectomy.

Differential detection of viral DNA and RNA in situ in cells infected with human cytomegalovirus.

We have analyzed the ability to use in situ cytohybridization to distinguish between human cytomegalovirus (HCMV) DNA and RNA in human cells infected in vitro. Two different viral-specific probes were used, one for an abundantly expressed late gene, and one which includes at least two genes coding for immediate early (IE) proteins. In productively infected cells, hybridization of the late gene probe extended over both the nucleus and cytoplasm and was RNase sensitive, whereas hybridization of the IE probe was restricted to the nucleus and was DNase-sensitive. In nonproductively infected cells hybridization of the IE probe was localized to the cytoplasm and was RNase-sensitive. The specific nuclease sensitivities indicate that a cytoplasmic hybridization pattern correlates with detection of viral RNA sequences, whereas a nuclear pattern represents detection of viral DNA. These results demonstrate that in situ cytohybridization can potentially be used to determine the extent of HCMV infection in a particular tissue or cell type by distinguishing between transcription and replication of specific viral genes.

Viral infections associated with bone marrow transplantation.

Bone marrow transplantation is complicated by a sequential occurrence of viral infections, the predictability of which influences disease management. Among these infections are herpes simplex virus, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, respiratory viral infections, hepatitis viral infections, and gastrointestinal infections. The approach to the treatment and prevention of these infections is discussed.

Cytomegalovirus infection of the fetus and neonate.

Human CMV infection, the most common virus infection of the fetus and neonate, can occur in utero as "congenital infection" or during the first weeks of life as "perinatal infection." Congenital infection presents either as clinically apparent or as silent infection. Perinatal CMV infection is a common form of CMV acquisition and produces minimal, if any, disease in the full-term infant. The relative occurrence of the viral syndromes in the population and means to prevent this infection are discussed.