Disease In AIDS Patients Research Articles

The Novel Lambda-Interferons IL-28A and IL-29 Mediate Proinflammatory, Antiproliferative, and Antiviral Signals in Intestinal Epithelial Cells

Background: Human cytomegaly virus (HCMV) is a ubiquitous virus that is a major cause of morbidity and mortality in immunocompromised individuals. During severe HCMV disease in AIDS patients, the gastrointestinal tract is frequently involved and typically shows mucosal lesions resembling features of inflammatory bowel disease. Recently a novel group of cytokines (IL-28A/B and IL-29, also termed interferon/IFN-λs), which signal through a receptor complex consisting of IL-28R and IL-10R2, was described. Aim: Given the functional similarity of these cytokines to type I IFNs, we analyzed IFN-λ receptor expression, signal transduction, and specific biological functions including antiviral properties against HCMV of this cytokine system in intestinal epithelial cells (IEC). Methods: Expression of IL-28R and IL-10R2 mRNA in IEC lines was analyzed by RT-PCR. Signal transduction was studied by Western blotting and ELISA. The induction of antiviral proteins was investigated by semiquantitative PCR. Cell proliferation was measured by MTS assay. Fas induced apoptosis was analyzed by flow cytometry. Antiviral properties of IL-28A and IL-29 were studied in HCT116 cells infected with HCMV. Infection was followed by staining cells with an antibody against HCMV immediate early protein (72 kD). Results: The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IFN-λ receptor subunits IL-28R and IL-10R2. IL-28A and IL-29 binding to its receptor complex activates ERK-1/2 and SAPK/JNK MAP kinases, and Akt resulting in increased IL-8 protein expression. IFN-λ also induced phosphorylation of STAT1 and significantly increased the mRNA expression of SOCS-3 and the antiviral proteins MxA and 2′,5′-OAS. These signals resulted in an up to 80% reduction of cells positive for HCMV immediate early protein after HCMV infection. Both, IL-28A and IL-29 decreased significantly cell proliferation (P < .005 and P < .05, respectively) but had no effect on Fas-induced apoptosis. Conclusions: IEC express functional receptors for IFN-λ, which mediate proinflammatory, antiviral, and antiproliferative signals in IEC suggesting a potential for therapeutic use in certain viral infections and as (antiproliferative) anti-cancer therapy.

Therapeutic strategies for human microsporidia infections

Over the past 20 years, microsporidia have emerged as a cause of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers and the elderly. Enterocytozoon bieneusi and the Encephalitozoon spp., Encephalitozoon cuniculi, Encephalitozoon hellem and Encephalitozoon intestinalis, are the most frequently identified microsporidia in humans, and are associated with diarrhea and systemic disease. The microsporidia are small, single-celled, obligately intracellular parasites that have been identified in water sources, as well as in wild, domestic and food-producing farm animals, thereby raising concerns for waterborne, foodborne and zoonotic transmission. Current therapies for microsporidiosis include albendazole, a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon spp., although it is less effective against Encephalitozoon bieneusi. Fumagillin, an antibiotic and antiangiogenic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and E. bieneusi; however, is toxic when administered systemically to mammals. Recent studies are also focusing on compounds that target the microsporidia polyamines (e.g., polyamine analogs), methionine aminopeptidase 2 (e.g., fumagillin-related compounds), chitin inhibitors (e.g., nikkomycins), topoisomerases (e.g., fluoroquinolones) and tubulin (e.g., benzimidazole-related compounds).

Microsporidiosis: An emerging and opportunistic infection in humans and animals

Microsporidia have emerged as causes of infectious diseases in AIDS patients, organ transplant recipients, children, travelers, contact lens wearers, and the elderly. These organisms are small single-celled, obligate intracellular parasites that were considered to be early eukaryotic protozoa but were recently reclassified with the fungi. Of the 14 species of microsporidia currently known to infect humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common causes of human infections and are associated with diarrhea and systemic disease. Species of microsporidia infecting humans have been identified in water sources as well as in wild, domestic, and food-producing farm animals, raising concerns for waterborne, foodborne, and zoonotic transmission. Current therapies for microsporidiosis include albendazole which is a benzimidazole that inhibits microtubule assembly and is effective against several microsporidia, including the Encephalitozoon species, but is less effective against E. bieneusi. Fumagillin, an antibiotic and anti-angiogenic compound produced by Aspergillus fumigatus, is more broadly effective against Encephalitozoon spp. and Enterocytozoon bieneusi but is toxic when administered systemically to mammals. Gene target studies have focused on methionine aminopeptidase 2 (MetAP2) for characterizing the mechanism of action and for identifying more effective, less toxic fumagillin-related drugs. Polyamine analogues have shown promise in demonstrating anti-microsporidial activity in culture and in animal models, and a gene encoding topoisomerase IV was identified in Vittaforma corneae, raising prospects for studies on fluoroquinolone efficacy against microsporidia.

Enhanced Killing ofCandida albicansby Human Macrophages Adherent to Type 1 Collagen Matrices via Induction of Phagolysosomal Fusion

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

Low prevalence of Helicobacter pylori but high prevalence of cytomegalovirus‐associated peptic ulcer disease in AIDS patients: Comparative study of symptomatic subjects evaluated by endoscopy and CD4 counts

The role of Helicobacter pylori infection in gastroduodenal lesions might be different between the general population and AIDS patients. The aim of the present study was to compare the prevalence of H. pylori and cytomegalovirus (CMV) infection in AIDS patients and HIV-negative controls. The impact of CD4 lymphocyte counts on H. pylori and CMV infection in the same subjects was also assessed. One hundred and fifty-six patients (52 HIV-positive, 104 HIV-negative) with gastrointestinal symptoms were evaluated with upper gastrointestinal endoscopy and biopsy. Comparison of the prevalence of H. pylori and CMV infection was made by dividing AIDS patients into two groups: those with CD4 counts >100/mm3 and those with CD4 counts <100/mm3, and ulcer and non-ulcer patients. In comparison with HIV-negative controls, AIDS patients had a lower prevalence of H. pylori infection (P < 0.0001) but a higher prevalence of CMV infection (P < 0.0001). Cytomegalovirus infection was frequently found in AIDS patients with CD4 count <100/mm3, in comparison with those with a CD4 count >100/mm3. In AIDS patients, CMV was more frequently detected in subjects with peptic ulcers (P = 0.0125). Conversely, the prevalence of H. pylori infection in AIDS patients was not different between those with and without peptic ulcers. The low prevalence of H. pylori infection and peptic ulcer in AIDS patients suggests a different role of H. pylori infection in peptic ulcer or even a different mechanism of peptic ulcerogenesis in HIV-positive subjects. Cytomegalovirus, rather than H. pylori, may be the main causative pathogen of peptic ulcers in AIDS patients.

Cytomegalovirus glycoprotein B genotypes and central nervous system disease in AIDS patients.

To investigate any association between cytomegalovirus glycoprotein B (CMV gB) subtypes and central nervous system (CNS) disease in AIDS patients, proportions of different gB genotypes detected in AIDS patients with CNS disease were compared with the gB genotypes detected in AIDS patients with no neurological disorder. The patients were matched by CD4+ cell counts. CMV was detected by PCR in cerebrospinal fluid (CSF) samples obtained from AIDS patients with CNS disease and from urine and saliva samples obtained from AIDS patients without CNS disease. CMV strains obtained were digested by restriction enzymes HinffI and RsaI to classify the genotypes. The CMV gB genotype was determined in 26 CSF samples. Of these, 11/26 (42.3%) typed as gB group 1, seven (26.9%) as gB2, four (15.4%) as gB3, and four (15.4%) as gB4. The CMV gB genotype frequency distribution in the 42 AIDS patients without CNS disease showed that 18/42 (42.8%) were classified as gB group 1, 10 (23.8%) as gB2, seven (16.6%) as gB3, and seven (16.6%) as gB4. In the present study, no association was found between CMV gB genotypes and CMV-related central nervous system disease.

Escherichia coli enterovirulent phenotypes in Zambians with AIDS-related diarrhoea

Persistent diarrhoea is a major cause of morbidity and mortality in AIDS patients, and consequently animportant public health problem in sub-Saharan Africa. Although intestinal protozoa and bacteria are detected in many of these patients, a substantial proportion of disease remains unexplained even after intensive investigation. HEp-2 cell adherent Escherichia coli have been described in AIDS patients with persistent diarrhoea, but their contribution to the overall burden of disease is not yet defined. We studied HEp-2 cell adherence of E. coli isolates from 116 adult Zambian AIDS patients and 153 healthy controls obtained in 1993 or 1998–99. Enteroaggregative, enteropathogenic, and diffusely adherent phenotypes were observed in E. coli isolates from both AIDS patients and controls, but cytotoxic phenotypes were only isolated from the AIDS patients. There was no evidence of seasonality in the frequency of isolation, and there was no evidence of long-term carriage. Light and electron microscopy of distal duodenal biopsies did not reveal any bacteria closely associated with the brush border. Isolates were less susceptible to amoxycillin, tetracycline, and sulfonamides than to newer antibiotics. Enterovirulent E. coli appear to contribute to intestinal disease in AIDS patients in Zambia but asymptomatic carriage is common. Antibiotic trials should be carried out.

Human cytomegalovirus resistance to antiviral drugs: diagnosis, monitoring and clinical impact.

The incidence of human cytomegalovirus (HCMV) disease in AIDS patients decreased dramatically after the introduction, a few years ago, of highly active antiretroviral combination therapy. As a consequence, the emergence of drug-resistant HCMV strains is no longer a major problem in HIV-infected individuals. However, HCMV resistance to antiviral drugs is presently recognized as an emerging problem in transplantation settings. The mechanisms of HCMV drug resistance will be analysed along with the clinical features relevant to the emergence of drug-resistant HCMV strains during antiviral treatment of patients receiving either solid organ or haematopoietic stem cell transplantation.

Interleukin-2 Immunotherapy of Murine Cytomegalovirus Retinitis during MAIDS Correlates with Increased Intraocular CD8+ T-Cell Infiltration

AIDS-related human cytomegalovirus retinitis continues to be an important sight-threatening disease in AIDS patients who do not respond to highly active antiretroviral therapy. We have shown previously that systemic cytokine immunotherapy with interleukin-2 (IL-2) will protect against experimental murine cytomegalovirus (MCMV) in mice with a murine retrovirus-induced immunodeficiency syndrome (MAIDS). Since IL-2 serves as a Th1 immunoregulatory cytokine, we hypothesized that IL-2-induced protection against MCMV retinitis during MAIDS would correlate with a measurable increase in the number of natural killer (NK) cells and/or CD8+ T cells that infiltrate the eye in response to MCMV infection of the retina. We therefore performed a study to quantify and compare the number of NK cells and CD8+ T cells that infiltrate MCMV-infected eyes in untreated and IL-2-treated mice with MAIDS at 3 days and 5 days after subretinal MCMV inoculation. Double-label flow cytometric analysis revealed the detection of measurable numbers of both NK cells and CD8+ T cells in MCMV-infected eyes of untreated MAIDS mice destined to develop retinitis. In contrast, IL-2 immunotherapy during MAIDS correlated with a 10-fold increase by day 5 after inoculation in the number of CD8+ T cells in MCMV-infected eyes destined to be resistant to retinitis. However, IL-2 immunotherapy during MAIDS had no appreciable effect on the number of NK cells that infiltrated MCMV-infected eyes. Taken together, our findings suggest that function of cytotoxic lymphocytes that infiltrate the eye may be more important than absolute numbers of cytotoxic lymphocytes that infiltrate the eye when assessing the protective effects of IL-2 immunotherapy on MCMV retinitis during MAIDS.

Detection of late pp67-mRNA by NASBA in peripheral blood for the diagnosis of human cytomegalovirus disease in AIDS patients.

To evaluate the value of late-pp67-mRNA nucleic acid sequence-based amplification (NASBA) in comparison to DNA-PCR, blood culture and pp65-antigenemia assay for the detection of human cytomegalovirus (HCMV) disease in HIV-infected patients. The results of pp67-mRNA NASBA, DNA-PCR, culture and pp65-antigenemia assay were compared in 402 whole blood specimens of 98 HIV-infected patients with a low CD4 lymphocyte count who had not yet received highly active antiretroviral therapy (HAART). Thirty-seven samples were obtained from 30 patients with a diagnosis of HCMV disease and 365 samples from 68 patients without HCMV disease. The highest agreement of test results was observed between pp67-mRNA NASBA and quantitative pp65-antigenemia, with a threshold of nine antigen-positive cells/10(5) leukocytes (kappa-value 0.70, 95% CI=0.58-0.82). The sensitivity of pp67-mRNA NASBA for the diagnosis of HCMV disease (59.3%) was identical to that of the quantitative pp65-antigenemia assay, higher than that of the blood culture (48.2%) but lower than that of the DNA-PCR (77.8%). Pp67-mRNA NASBA (92.3%), quantitative pp65-antigenemia assay (92.3%) and blood culture (93.9%) were highly specific for the diagnosis of HCMV disease and as a result, had a higher positive predictive value (76.2, 76.2 and 76.5%, respectively) than the qualitative DNA-PCR (58.3%) and the qualitative pp65-antigenemia assay (47.6%). pp67-mRNA NASBA, an easy and rapid to perform assay, well-standardised by virtue of co-amplified internal system control RNA, provides a high specificity and positive predictive value for the diagnosis of HCMV disease in HIV-infected patients, comparable to that of the quantitative pp65-antigenemia assay and blood culture.

Multicenter Brazilian Study of Oral Candida Species Isolated from Aids Patients

Oropharyngeal candidiasis continues to be considered the most common opportunistic disease in Aids patients. This study was designed to investigate species distribution, serotype and antifungal susceptibility profile among Candida spp. isolated from the oral cavity of Aids patients recruited from six Brazilian university centers. Oral swabs from 130 Aids patients were plated onto CHROMagar Candida medium and 142 isolates were recovered. Yeast isolates were identified by classical methods and serotyped using the Candida Check or target system-Iatron. Antifungal susceptibility testing was performed according to the NCCLS microbroth assay. C. albicans was the most frequently isolated species (91%), and 70% of the isolates belonged to serotype A. We detected 12 episodes of co-infection (9%), including co-infection with both serotypes of C. albicans. Non-albicans species were isolated from 12 episodes, 50% of them exhibited DDS or resistance to azoles. Otherwise, only 8 out 130 isolates of C. albicans exhibited DDS or resistance to azoles. Brazilian Aids patients are infected mainly by C. albicans serotype A, most of them susceptible to all antifungal drugs.

CMV disease in AIDS patients: incidence of CMV disease and relation to survival in a population-based study from Oslo.

CMV disease is an important cause of morbidity and mortality in patients with AIDS. The purpose of this study was to investigate the incidence of CMV disease in a well-defined population of AIDS patients with a high rate of autopsy. No such study has previously been published from Scandinavia. A total of 248 patients who developed clinical AIDS in Oslo during the period 1 January, 1983 to 31 December, 1995 were included. Autopsy was performed in 152 of 213 deaths (71.3%). CMV disease was diagnosed in 95 patients. In the autopsy group, 73 patients (48%) had CMV disease, and in 52 of these patients CMV disease was first detected at autopsy. Retinitis was the most frequent manifestation, followed by adrenalitis, pneumonitis, encephalitis and gastrointestinal disease. No intravenous drug users (IVDUs) were diagnosed alive with CMV disease. All patients diagnosed with CMV disease before death had evidence of CMV disease at autopsy despite anti-CMV treatment. CMV disease was associated with increased risk of death. We conclude that CMV disease was frequent in patients with AIDS during the study period, was associated with increased mortality and was often diagnosed too late for the administration of appropriate therapy.

Phenotypic and genomic analyses of the Mycobacterium avium complex reveal differences in gastrointestinal invasion and genomic composition.

Mycobacterium avium and Mycobacterium intracellulare are closely related organisms and comprise the Mycobacterium avium complex. These organisms share many common characteristics, including the ability to cause life-threatening respiratory infections in people with underlying lung pathology or immunological defects and occasionally in those with no known predisposing conditions. However, the ability to invade the mucosa of the gastrointestinal tract and cause disseminated disease in AIDS patients has not been epidemiologically linked to M. intracellulare and appears to be unique to M. avium. We compared the abilities of M. avium and M. intracellulare to tolerate the acidic conditions of the stomach, to resist the membrane-disrupting activity of cationic peptides, and to invade intestinal epithelial cells in vitro and in vivo. We observed that M. avium and M. intracellulare were both tolerant to the acidic conditions encountered in the stomach and resistant to cationic peptides. However, when strains of M. avium and M. intracellulare were examined for their ability to enter cultured human intestinal cells or mouse intestinal mucosa, we observed that M. avium could invade more efficiently than M. intracellulare. To elucidate the basis of this pathogenic difference and identify genes involved in the invasion of the intestinal mucosa, we performed chromosomal DNA subtractive hybridization using M. avium and M. intracellulare chromosomal DNAs. In all, 21 genes that were present in M. avium but absent in M. intracellulare were identified, including some that may be associated with the ability of M. avium to invade the intestinal mucosa.

Candida albicans is phagocytosed, killed, and processed for antigen presentation by human dendritic cells.

Candida albicans is a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranes of the healthy host. Candida is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients, and of oropharyngeal disease in AIDS patients. As the induction of cell-mediated immunity to Candida is of critical importance in host defense, we sought to determine whether human dendritic cells (DC) could phagocytose and degrade Candida and subsequently present Candida antigens to T cells. Immature DC obtained by culture of human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 phagocytosed unopsonized Candida in a time-dependent manner, and phagocytosis was not enhanced by opsonization of Candida in serum. Like macrophages (Mphi), DC recognized Candida by the mannose-fucose receptor. Upon ingestion, DC killed Candida as efficiently as human Mphi, and fungicidal activity was not enhanced by the presence of fresh serum. Although phagocytosis of Candida by DC stimulated the production of superoxide anion, inhibitors of the respiratory burst (or NO production) did not inhibit killing of Candida, even when phagocytosis was blocked by preincubation of DC with cytochalasin D. Further, although apparently only modest phagolysosomal fusion occurred upon DC phagocytosis of Candida, killing of Candida under anaerobic conditions was almost equivalent to killing under aerobic conditions. Finally, DC stimulated Candida-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of both viable and heat-killed Candida cells. These data suggest that, in vivo, such interactions between DC and C. albicans may facilitate the induction of cell-mediated immunity.

Compromiso pulmonar por linfoma no Hodgkin diseminado en un paciente con SIDA

Pulmonary involvement due to disseminated non Hodgkin lymphoma (LNH), is an unusual cause of lung disease in AIDS patients. We report a 38 years old male patient, with advanced AIDS, who, in the course of three weeks, developed cough, dyspnea and fever. The chest X ray film showed diffuse thickening of the peribronchovascular connective tissue with possible mediastinal lymph node enlargement. The evolution was unfavorable with hypoxemia, severe anemia, liver damage and elevated levels of lactic dehydrogenase. The presumptive initial diagnoses were Pneumocystis carinii pneumonia, pulmonary tuberculosis with hematogenous dissemination and Kaposi sarcoma. Definitive diagnosis was made through a transbronchial biopsy performed the day before his death. The pathological and inmunohistochemical report demonstrated a highly aggressive lymphoma (lymphoblastic, B precursor). This finding was confirmed by autopsy that revealed multiple organ involvement.

Cryptosporidium parvum genome project

A lack of basic understanding of parasite biology has been a limiting factor in designing effective means of treating and preventing disease caused by Cryptosporidium parvum. Since the genomic DNA sequence encodes all of the heritable information responsible for development, disease pathogenesis, virulence, species permissiveness and immune resistance, a comprehensive knowledge of the C. parvum genome will provide the necessary information required for cost-effective and targeted research into disease prevention and treatment. With the recent advances in high-throughput automated DNA sequencing capabilities, large-scale genomic sequencing has become a cost-effective and time-efficient approach to understanding the biology of an organism. In addition, the continued development and implementation of new software tools that can scan raw sequences for signs of genes and then identify clues as to potential functions, has provided the final realization of the potential rewards of genome sequencing. To further our understanding of C. parvum biology, we have initiated a random shotgun sequencing approach to obtain the complete sequence of the IOWA isolate of C. parvum. Our progress to date has demonstrated that sequencing of the C. parvum genome will be an efficient and costeffective method for gene discovery of this important eukaryotic pathogen. This will allow for the identification of key metabolic and immunological features of the organism that will provide the basis for future development of safe and effective strategies for prevention and treatment of disease in AIDS patients, as well as immunocompetent hosts. Moreover, by obtaining the complete sequence of the C. parvum genome, effective methods for subspecific differentiation (strain typing) and epidemiologic surveillance (strain tracking) of this pathogen can be developed.

Microsporidial AIDS cholangiopathy due to Encephalitozoon intestinalis: case report and review

Microsporidia are increasingly recognized as opportunistic infections in immunodeficient patients, predominantly patients with AIDS. The two microsporidia most commonly associated with disease in AIDS patients are Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously known as Septata intestinalis). The most common clinical presentation of microsporidiosis in AIDS patients is diarrhea, most commonly caused by the Enterocytozoon bieneusi species. Encephalitozoon intestinalis is a recently described species that has been reported to cause disseminated human infection including cholangitis. We report a case of AIDS cholangiopathy that presented with abdominal pain and cholestatic liver tests. Ultrasound examination and ERCP revealed a picture of sclerosing cholangitis. Bile samples obtained at ERCP were negative for microsporidia; stool studies for microsporidia and cryptosporidia were also negative. No organisms were identified on routine light microscopy of the biopsy specimens from the duodenum, ampulla, and bile duct. E. intestinalis spores were demonstrated in the bile duct biopsies, by methylene blue and azure 11 staining and confirmed by electron microscopy. Albendazole therapy was successful in eradicating E. intestinalis with clinical improvement and improvement in CD4 count. However, the cholangiographic picture did not improve and repeat cholangiography revealed progressive bile duct injury. Albendazole therapy was delayed and may have been too late to prevent bile duct damage; the drug had to be approved by the US Food and Drug Administration for compassionate use. This is an unusual case of sclerosing cholangitis caused by an unusual organism and requiring biliary sphincterotomy and stent placement for progressive stricturing despite eradication of the infection.

Generation of a Polyclonal Fab Phage Display Library to the Protozoan Parasite Cryptosporidium parvum

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.

Human cytomegalovirus: challenges, opportunities and new drug development.

In the age of highly active antiretroviral therapy, the incidence of human cytomegalovirus (HCMV) retinitis in AIDS patients has decreased substantially. However, this change does not indicate that HCMV disease in AIDS patients and other immunocompromised patients has abated and is no longer a concern. On the contrary, HCMV disease in graft recipients, newborns, and even in AIDS patients still accounts for considerable morbidity, and drug resistance to the anti-HCMV compounds is a major problem. Furthermore, HCMV may have a role in metabolic diseases, such as atherosclerosis. Fortunately there are novel and potentially very effective new compounds undergoing pre-clinical and clinical evaluation. These developments point the way toward new therapies and also to a clearer understanding of the biology of HCMV replication, infection and disease.

Protection against establishment of retroviral persistence by vaccination with a live attenuated virus.

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.