Immunophenotypic characterization of peripheral T lymphocytes in Mycobacterium tuberculosis infection and disease.

The cellular immune response probably plays a pivotal role in determining the clinical outcome after exposure to Mycobacterium tuberculosis. We used multi-parameter flow-cytometry to evaluate the distribution of T-lymphocyte subsets during infection and disease caused by M. tuberculosis. Samples were obtained from 71 volunteers to identify the T CD4+ and CD8+ lymphocyte numbers, and the activation plus memory/naïve phenotypes, as defined by CD38, HLA-DR, CD45RA and CD27 markers. Subjects were divided into 18 healthy volunteers without detectable reaction to purified protein derivative (PPD-), 18 health care workers with a recent conversion to PPD, 20 patients with active pulmonary tuberculosis (TBC) and 15 patients with treated TBC at 6 months of therapy. By multiple-comparison analyses, the T CD4+ lymphocyte number of the TBC group was lower than the PPD- group (P < 0.05). This difference was apparently lost after treatment. The higher and the lower number of naïve T CD4+ cells was observed in the PPD- and TBC group, respectively. CD8+ T lymphocytes were also statistically different among the four groups (P = 0.0002), lower in the TBC group (P < 0.05). CD8+ T lymphocyte activation was evaluated by the CD38 and HLA-DR surface expression. The percentage distribution of these markers was statistically different between the four groups (P = 0.0055). TBC patients had a higher percentage of CD38+ cells and mean fluorescence index, suggesting an overall increase of cell activation. These results suggest that peripheral T lymphocytes reflect cellular activation during TBC, along with possible redistribution of naïve, memory/effector and late differentiated memory/effector phenotypes in the peripheral blood after infection and disease caused by M. tuberculosis.

A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers.

Viruslike particles (VLPs) produced from the L1 protein of several papillomaviruses have induced protection from infection after live challenge in animal models. In the present study, the safety and immunogenicity of a human papillomavirus (HPV)--11 L1 VLP candidate vaccine were measured in a phase 1, dose-finding trial in humans. The vaccine was well tolerated and induced high levels of both binding and neutralizing antibodies. Marked increases in lymphoproliferation to HPV--11 L1 antigens were noted after the second vaccination. In addition, lymphoproliferation was induced after vaccination in peripheral blood mononuclear cells (PBMC) stimulated with heterologous L1 VLP antigens of HPV types 6 and 16. Statistically significant increases in HPV antigen--specific interferon--gamma and interleukin-5 production were measured from PBMC culture supernatants. This candidate HPV VLP vaccine induced robust B and T cell responses, and T cell helper epitopes appear to be conserved across HPV types.

Highly active antiretroviral therapy results in a decrease in CD8+ T cell activation and preferential reconstitution of the peripheral CD4+ T cell population with memory rather than naive cells.

OBJECTIVE: Highly active antiretroviral therapy (HAART) can produce marked increases in peripheral blood CD4+ T cells and decreases in HIV plasma RNA copy numbers. However, it is not clear whether these absolute changes will be accompanied by a recovery in the known naive CD4+ T cell depletion or a decrease in the marked CD8+ T cell activation. DESIGN: Twenty-nine patients were enrolled in studies of either nucleoside therapy alone or nucleoside therapy combined with a protease inhibitor (zidovudine + lamivudine + indinavir). One hundred and ninety-one examinations were carried out at three baseline time points and during 40 weeks of follow-up to evaluate the effect of HAART on CD4+ memory/naive phenotype and CD8+ T cell activation. METHODS: CD4+ and CD8+ T cell number, CD62L/CD45RA expression on CD4+ T cells and CD38 expression on CD8+ T cells were measured by three-color flow cytometry. RESULTS: Most protease inhibitor treated patients had a significant rise in CD4+ numbers. The marked rise in the CD4+ T cells seen in individuals in this study was not accompanied over a 40-week period by a change in the abnormally low CD4+ naive compartment, and thus was almost completely of memory phenotype. The CD38 expression on CD8+ cells fell during treatment, and decreased to a greater degree than the comparable rise in CD4+ T cell counts. This decrease continued in many patients after the CD4+ T cell rise or viral load decline had plateaued. CONCLUSION: HAART results in changes in activation to a greater extent than absolute changes in CD4+ T cell numbers, but is not accompanied by an increase in naive CD4+ T cells. Measurements of CD4+ T cell numbers alone may not allow appropriate interpretation of immune activation or immune competence in patients receiving those drugs.

Isolation and propagation of human papillomavirus type 16 in human xenografts implanted in the severe combined immunodeficiency mouse.

We report the isolation and propagation of human papillomavirus type 16, the main agent of cervical cancer, using human foreskin fragments implanted in severe combined immunodeficiency mice. The infection produced viral particles, and with each passage of the virus it caused lesions identical to intraepithelial neoplasia, the precursor to carcinoma.

In vitro infection and type-restricted antibody-mediated neutralization of authentic human papillomavirus type 16.

Human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Studies of model systems with animal papillomaviruses have demonstrated the importance of neutralizing antibodies in preventing papillomavirus-associated disease. The assessment of neutralizing antibody responses against HPV-16, previously hampered by the lack of a viral source, was enabled by the recent propagation of an HPV-16 stock in xenografted severe combined immunodeficiency (SCID) mice. HPV-16 infection of an immortalized human keratinocyte cell line was demonstrated by detection of an HPV-16-specific spliced mRNA amplified by reverse transcriptase PCR. Infection was blocked by preincubation of the virus with antiserum generated against HPV-16 virus-like particles (VLPs) composed of the major capsid protein, L1. To examine potential cross-neutralizing activity among the different genital HPV types, rabbit antisera to L1 VLPs corresponding to HPV-6, -11, -18, -31, -33, -35, -39, and -45 were assayed for the ability to block the HPV-16 infection of cultured cells. Antiserum raised against HPV-33 L1 VLPs was the only heterologous antiserum which inhibited HPV-16 infection. Thus, a neutralization assay for HPV-16 may help to characterize the components required to compose a broadly efficacious genital HPV vaccine.

A randomized, double-blind trial of parenteral low dose versus high dose interferon-beta in combination with cryotherapy for treatment of condyloma acuminatum.

Forty-nine subjects were enrolled in a study comparing two dosages of parenterally administered interferon (IFN)-beta in combination with cryotherapy for the treatment of anogenital warts. Subjects were randomized to receive subcutaneous injections of either 2 x 10(6) or 4 x 10(6) IU/m2 of IFN-beta (Biogen) three times a week for a total of 6 weeks. Cryotherapy was administered concomitantly by aerosolization of liquid nitrogen at 10-day intervals. Systemic side- effects were modest in intensity and included fever, chills, myalgia, and headaches (flu-like symptoms). During the first 2 weeks of therapy, they were more common in the high dose group than in the low dose group (P = 0.02). Using survival analysis, there was no significant difference between the two groups in rates of resolution of warts present at baseline (P = 0.62). However, the rate of new lesion formation during the study was significantly lower in the high dose group (P = 0.04).

Therapeutic efficacy and complications of excisional biopsy of condyloma acuminatum.

BACKGROUND AND OBJECTIVES: Among the various treatment modalities for condyloma acuminatum, excisional cold-blade surgery appears excellent but it has been little studied and little used, particularly for lesions not located in the perianal area. GOALS: To examine the efficacy and complications of scissors excision of single anogenital warts. STUDY DESIGN: Retrospective analysis of single warts completely excised with scissors for the purpose of biopsy before patient entry in a randomized, placebo-controlled study of the efficacy and safety of various parenteral interferons in combination with cryotherapy. RESULTS: Of 152 patients entered in the main study, 85 patients were available for analysis. At 4 and 16 weeks after excision, 16 of 85 (19%) and 14 of 68 (21%) of the excised lesions recurred. After at 6 least months of follow-up, 2 of 11 (18%) of the excision sites demonstrated some evidence of pigmentation changes. CONCLUSIONS: Scissors excision of single anogenital warts has a high rate of success and acceptable long-term side-effects.

Neisseria elongata subsp. elongata, a case of human endocarditis complicated by pseudoaneurysm.

We report an unusual case of endocarditis caused by Neisseria elongata subsp. elongata. The illness was complicated by a ruptured mycotic aneurysm of the right brachial artery, with compression of the brachial plexus. A cure was achieved after aneurysm resection and treatment with intravenous ceftriaxone and gentamicin.

A randomized, double-blind, placebo-controlled trial of systemically administered interferon-alpha, -beta, or -gamma in combination with cryotherapy for the treatment of condyloma acuminatum.

One hundred fifty-two patients were enrolled in a study to evaluate 3 interferon (IFN) preparations used in combination with cryotherapy for treatment of anogenital warts. Subjects received subcutaneous injections (2 x 10(6) units/m2) of IFN-alpha n1, -beta, -gamma or placebo 3 times a week for 6 weeks and cryotherapy with liquid nitrogen. Subjects were followed < or = 1 year. Among patients followed > or = 12 weeks, two-thirds had a complete response. No significant differences in rates of complete response (P = .37) or reappearance of a wart at the initial site (P = .20) were noted among the treatment groups. However, patients who received IFN-beta or -gamma developed new warts at a significantly lower frequency (P = .02). IFN administration was associated with side effects but was well tolerated. IFN-beta was the least toxic of the 3 preparations and had the best therapeutic ratio.

Serological differentiation of human papillomavirus types 11, 16 and 18 using recombinant virus-like particles.

The L1 major capsid protein-coding sequences of human papillomavirus (HPV) types 11, 16 and 18 were expressed in the baculovirus system. Virus-like particles (VLPs) were purified from recombinant-infected Spodoptera frugiperda Sf9 cells and cell-free culture supernatants. Rabbits immunized with purified VLPs developed antibodies that reacted only with the specific VLP type used as the immunogen. In addition, rabbit antibodies raised against infectious HPV-11 virions only reacted with HPV-11 L1 VLPs and not with VLPs derived from either HPV-16 or HPV-18. These results suggest that HPV-11, HPV-16 and HPV-18 virions are antigenically distinct from one another. This observation should be considered in future studies of immune responses to HPV.

Human papillomavirus (HPV) type 11 recombinant virus-like particles induce the formation of neutralizing antibodies and detect HPV-specific antibodies in human sera.

Recombinant human papillomavirus type 11 (HPV-11) virus-like particles (VLPs) were tested for their ability to induce the formation of neutralizing antibodies, and were also tested for serodiagnostic capabilities in an ELISA in comparison with HPV-11 whole virions. VLPs, purified by CsCl density gradient centrifugation from the cell-free supernatant of Ac11L1-infected Sf9 suspension cell cultures, were used to immunize rabbits and anti-VLP antibodies were tested in the athymic mouse model of HPV-11 infection. Pretreatment of infectious HPV-11 virions with the immune serum of VLP-treated animals caused a marked reduction of graft growth (P < 10(-4)) and viral gene expression (P < 10(-4)), similar to the effects obtained using whole virion postimmune serum, and consistent with immune neutralization. To assess the serodiagnostic capabilities of VLPs, a VLP ELISA was developed and used to analyse sera that were tested previously in an HPV-11 whole virion ELISA. Specific antibodies were detected in 49% of patients' sera (P = 2 x 10(-4)), and individual VLP seroreactivities correlated with those previously obtained using whole virions as the antigen (r = 0.87; P < 10(-6)). These results indicate that recombinant VLPs can be used to elicit a neutralizing antibody response, and can substitute faithfully for native virions in the development of HPV-serodiagnostic immunoassays.

Evaluation of temperature sensitivity of human papillomavirus type 11 by using the human xenograft severe combined immunodeficiency mouse model.

The temperature sensitivity of human papillomavirus type 11 was evaluated by using a human xenograft severe combined immunodeficiency mouse model. Incubation of the virus for 1 h at a temperature higher than 56 degrees C but lower than 72 degrees C was sufficient to inhibit the virally induced growth of infected human tissue. However, 100 degrees C was necessary to completely inactivate HPV type 11 genome expression.

Efficacy and safety of 0.5% podofilox solution in the treatment and suppression of anogenital warts.

PURPOSE: For the patient-administered treatment of anogenital warts, 0.5% podofilox (podophyllotoxin), one of the active compounds of podophyllin, has been shown to be more effective than the vehicle alone. This study was designed to evaluate the safety and efficacy of 0.5% podofilox treatment followed by prophylaxis. PATIENTS AND METHODS: A total of 103 patients were entered in stage 1 of the study. Stage 1 was an open label study, and patients self-administered 0.5% podofilox twice daily for 3 consecutive days per week for 4 weeks. A total of 100 patients remained available for efficacy and safety analyses. At the end of stage 1, patients who had a complete response proceeded to stage 2 of the study. Patients who had a 50% to 99% reduction in measured total wart area were offered cryotherapy every 10 days, up to 5 times. If cleared of warts, they were also entered into stage 2. A total of 57 patients were enrolled into stage 2, a double-blind, randomized, placebo-controlled prophylactic study of 0.5% podofilox self-administered once daily for 3 days per week for 8 weeks, on the sites of healed warts. A total of 45 patients in stage 2 were available for efficacy analysis. RESULTS: By the end of stage 1, 68% of the warts had disappeared, and 29 of 100 patients (29%) had a complete response. A total of 49 patients had a 50% or greater improvement in wart area and underwent cryotherapy. Rates of local side effects after 1 week of treatment were 57% for inflammation, 39% for erosion, 47% for pain, 48% for burning, and 44% for itching. However, these symptoms and signs were mostly mild to moderate in intensity and diminished over time. Therefore, overall treatment was well tolerated. In stage 2, only 4 of 21 patients (19%) in the podofilox group experienced a recurrence as opposed to 12 of 24 (50%) in the placebo group (P = 0.031). As in stage 1, the side effects were modest, and the drug was well tolerated. CONCLUSION: This study confirms the efficacy and good tolerance of 0.5% podofilox in the treatment of anogenital warts. It also establishes the safety and superior efficacy of patient-administered podofilox over the vehicle alone as prophylaxis against recurrence of lesions. Although long-term efficacy and tolerance remain to be established, podofilox appears to be a useful agent in the control of this disease.

Two E2 binding sites (E2BS) alone or one E2BS plus an A/T-rich region are minimal requirements for the replication of the human papillomavirus type 11 origin.

Human papillomaviruses (HPVs) cannot be propagated in vitro, but the DNA can be replicated transiently in an assay in the presence of two trans-acting viral proteins, E1 and E2. Using this assay, we have defined the minimal cis-acting elements of the origin of replication of HPV type 11. Most HPV genomes are conserved at the origin of replication, and the core contains three E2 binding sites (E2BS) surrounding an A/T-rich spacer region. The present results show that the minimal requirement for replication is either two E2BS alone or the A/T-rich region plus one E2BS; in the latter case the relative position of the E2BS is important. In all the studies, the presence of both E1 and E2 proteins was essential for replication, yet only the E2BS was required at the origin. We have shown that E1, E2, and the origin of replication containing an E2BS from a complex in vitro, and our data are consistent with a model in which E2 acts to target E1 to the HPV type 11 replication origin.

Propagation of human papillomavirus type 11 in human xenografts using the severe combined immunodeficiency (SCID) mouse and comparison to the nude mouse model.

We report propagation of human papillomavirus (HPV) type 11 in human xenografts in the severe combined immunodeficiency (SCID) mouse and compare this new animal model for HPV infection to the previously established athymic nude mouse model. HPV-11-infected foreskin fragments grafted under the renal capsule of SCID mice formed large epithelial cysts that had the histologic and immunocytochemical features of HPV infection. This infection was successfully passaged to nude mice. Viral particles that reacted to an antibody directed to HPV-11 virions were identified from samples recovered from the SCID and nude mice. Viral DNA sequence analysis confirmed that the passaged virus was HPV-11. In a comparative experiment of the nude mouse and SCID mouse models, the latter produced HPV-11-infected xenografts that were larger and more often positive for HPV by immunocytochemistry and presence of viral mRNA than those propagated in the former model. Finally, we observed that growth of HPV-11-infected foreskin fragments in the SCID mouse model is not restricted to the kidney as in the nude mouse, but also can occur in the subcutis and the peritoneum.

Penile intraepithelial neoplasia: clinical presentation and an analysis of the physical state of human papillomavirus DNA.

Forty-four men with penile intraepithelial neoplasia (PIN) and a matched control group of 88 men with condyloma acuminatum were evaluated in three centers studying anogenital human papillomavirus (HPV) infections. PIN and condyloma groups could not be distinguished on the basis of historical features or clinical presentation. Although PINs were more likely than condylomata to be pigmented (31/46 [67%] vs. 33/97 [34%], P < .001), 43% of PIN III were not pigmented, suggesting that pigmentation is not a sensitive indicator of high-grade PIN. HPV-16 infection, as determined by in situ hybridization, was closely associated with PIN III (0/24 PIN I contained HPV-16 vs. 12/13 PIN III, P < .001). Southern blot analysis demonstrated only episomal viral genomes, suggesting that integration is not an early event in penile neoplasia.