Therapeutic vaccination with papillomavirus E6 and E7 long peptides results in the control of both established virus-induced lesions and latently infected sites in a pre-clinical cottontail rabbit papillomavirus model.

This study was performed to test the therapeutic efficacy of overlapping long E6 and E7 peptides, containing both CD4+ T-helper and CD8+ CTL epitopes, on CRPV-induced lesions, which is an appropriate pre-clinical model for HPV diseases, including recurrent respiratory papillomatosis (RRP). Therapeutic peptide vaccination was able to significantly control wart growth (p < 0.01) and abrogate latent CRPV infection (p = 0.0006) compared to controls. Vaccination was associated with a T(H)1 T cell response, as suggested by a strong DTH skin test, antigen-specific proliferation of PBMC and a minimal IgG antibody response. Thus, this study shows promise for treatment of RRP by vaccination with long peptides.

Interaction of human papillomavirus type 11 E7 protein with TAP-1 results in the reduction of ATP-dependent peptide transport.

Human papillomaviruses (HPVs) cause benign and malignant epithelial tumors of the respiratory and genital mucosa. We previously reported that recurrent respiratory papillomas caused by HPV 6/11 express low levels of antibody-detectable TAP-1, the protein that transports peptides into the endoplasmic reticulum for assembly and presentation by MHC Class I, and that the extent of TAP-1 immunostaining is inversely related to the frequency of disease recurrence. We have now determined a mechanism for the reduction in TAP-1 detection. Anti-TAP-1 antibody immunoprecipitated very low amounts of protein from papilloma cells. However, immunoprecipitation of calreticulin, another member of the MHC I assembly complex, coprecipitated TAP-1 at levels comparable to those of uninfected cells. Immunoprecipitation of an HPV-positive cell line with either anti-TAP-1 or anti-calreticulin coprecipitated HPV E7 protein. Finally, purified HPV 11 E7 protein inhibited ATP-dependent peptide transport in vitro. We propose that the interaction of E7 with TAP-1 prevents TAP-1 antibody detection and efficient peptide transport, resulting in poor presentation of viral antigen on HPV-infected cells and thus failure to mount an effective immune-mediated prevention of disease recurrence.

Population pharmacokinetics of dapsone in children with human immunodeficiency virus infection.

BACKGROUND: Previous studies of dapsone pharmacokinetics in children have been too small to allow assessment of the relationships between dapsone pharmacokinetic parameters and patient characteristics or markers of efficacy and toxicity. METHODS: We used population analysis to estimate dapsone pharmacokinetic parameters in children participating in a phase I/II study of daily and weekly dapsone in children with human immunodeficiency virus (HIV) infection. With use of the program NONMEM and a 1-compartment open model, the influence of demographic and clinical characteristics on oral clearance (CL/F) and oral volume of distribution (V/F) were examined. Measures of drug exposure (area under the concentration-time curve [AUC] and predicted concentrations just before and 2 hours after administration) were estimated for each patient and correlated with markers of efficacy and toxicity. RESULTS: Sixty children (median age, 3 years; age range, 2 months to 12 years) contributed 412 dapsone concentrations collected after 175 study doses. Final parameter estimates were 1.40 L/kg for V/F, 0.0283 L/kg/h for CL/F, and 2.66 for the absorption rate constant. Of the clinical characteristics evaluated, dapsone CL/F was significantly increased by 50% in children taking rifabutin, by 39% in black children, and by 38% in children younger than 2 years old. Although no significant correlations were found between any dapsone exposure parameter and markers of toxicity, increased AUC was associated with a decreased risk of Pneumocystis carinii pneumonia (PCP). CONCLUSION: Ethnicity, age, and concomitant rifabutin use were associated with dapsone CL/F, with more rapid clearance observed in black children, children younger than 2 years old, and children receiving rifabutin. Dapsone pharmacokinetic parameters were not associated with toxicity, but higher dapsone AUC was associated with decreased risk of PCP. Monitoring of serum dapsone levels may be needed for optimal management of dapsone for PCP prophylaxis in children.

Altered expression of TAP-1 and major histocompatibility complex class I in laryngeal papillomatosis: correlation of TAP-1 with disease.

Recurrent respiratory papillomatosis (RRP) is an insidious disease caused by human papillomavirus (HPV) infection. It is characterized by a variable clinical course that can include frequent disease recurrence, significant morbidity, and occasional mortality. The mechanisms responsible for the variability in the clinical course and the persistence of latent HPV infection remain unknown. Effective T-cell-mediated clearance of HPV-infected cells may be defective in patients with RRP, leading to recurrent disease and failure to suppress latent HPV reactivation. This study describes the down-regulation of the transporter associated with antigen presentation (TAP-1) and the major histocompatibility complex (MHC) class I protein expression in laryngeal papilloma tissue biopsies and cell culture of primary explants. There was a statistically significant correlation between reduction of TAP-1 expression in biopsy tissues and rapid recurrence of disease. Patients with RRP had less frequent recurrence if their papillomas expressed TAP-1 at levels close to that of normal tissue, compared with those with very low expression of TAP-1, who had frequent recurrence (32 versus 5 weeks to the next surgical intervention). These findings suggest that HPV may evade immune recognition by down-regulating class I MHC cell surface expression via decreased TAP-1 levels. Expression of TAP-1 could be used for prognostic evaluation of disease severity. Gamma interferon was able to restore class I MHC expression at the surfaces of laryngeal papilloma cells in culture. This up-regulation of class I MHC antigen at the cell surface potentially allows the infected cell to become a target for the immune system again. This finding provides some promise for nonsurgical treatment of laryngeal papillomas.

Recurrent respiratory papillomatosis: altered CD8(+) T-cell subsets and T(H)1/T(H)2 cytokine imbalance.

Human papillomaviruses (HPVs) cause benign papillomas and squamous cell carcinomas in the genital and respiratory tracts. Recurrent respiratory papillomas (RRP) generate a high level of morbidity and significant mortality because of their location, resistance to treatment, and relentless recurrence that can vary in frequency in a given patient and between patients. We have found that T-cells from these patients, when exposed to or isolated from autologous papilloma tissue, have an elevated percentage of CD8(+), CD28(-) T-cells, and that T-cells from many of these patients express an increase in T(H)2-like cytokine mRNA in response to autologous papilloma tissue. Furthermore, both of these immunologic findings correlate with disease severity. These observations suggest that patients with RRP, and possibly others with refractory HPV-induced lesions, are unable to manage their disease with an appropriate and effective HPV-specific, T-cell response. This immune imbalance may be responsible for the development and severity of HPV-induced respiratory papillomatosis.

Pharmacokinetics of dapsone administered daily and weekly in human immunodeficiency virus-infected children.

Although dapsone is a commonly used alternative agent for prophylaxis against Pneumocystis carinii pneumonia in children intolerant to trimethoprim-sulfamethoxazole, there are few data that describe dapsone pharmacokinetics in children. We studied dapsone pharmacokinetics in 30 children (median age, 2.8 years; age range, 0. 3 to 12 years) receiving a new proprietary liquid preparation by three dosing regimens (1 mg/kg of body weight daily, 2 mg/kg daily, or 4 mg/kg weekly). Dosing of children with 2 mg/kg daily or 4 mg/kg weekly resulted in peak concentrations equivalent to those reached in adults receiving 100-mg tablets daily. For the entire population, the median half-life was 22.2 h (range, 7.1 to 40.3 h), the median oral clearance was 0.0365 liter/kg/h (range, 0.0104 to 0.1021 liter/kg/h), and the median oral apparent volume of distribution was 1.13 liters/kg (range, 0.50 to 2.32 liters/kg). The median dapsone oral clearance was significantly increased in those infants less than 2 years of age compared to the oral clearance in those over 2 years of age (0.0484 versus 0.0278 liter/kg/h; P = 0.011). These data suggest that absorption of this liquid preparation is adequate and that the concentrations in the sera of children receiving 2 mg/kg daily or 4 mg/kg weekly are equivalent to those seen in adults receiving standard dapsone dosing. Dapsone oral clearance appears to be increased in children under 2 years of age.

The specificity of synovial IgM rheumatoid factors (RF) for genetically engineered IgG antibodies is not affected by the method used to immortalize RF-producing B cells.

Previously, we have shown that some rheumatoid factors (RFs) produced by Epstein-Barr virus (EBV)-transformed B cells from patients with rheumatoid arthritis (EBV-RA-RF) appear to be disease-specific autoantibodies that bind differently to defined epitopes on genetically engineered IgG antibodies, compared with RFs expressed by patients with Waldenstrom's macroglobulinaemia (Wmac-RFs) and healthy immunized donors (HID-RFs). To exclude the possibility that EBV transformation is responsible for these differences, we have now studied 15 other monoclonal IgM RFs from patients with RA that were produced by heterohybridoma-B-cell fusion (HRA-RFs). These HRA-RFs show the same gross specificity profiles for IgG as do their EBV-RA-RF counterparts. However, when the specificities of the HRA-RF and EBV-RA-RF panels were combined and compared with those RFs from patients with Wmac or HID, significant differences in binding specificity were again observed. Hybrid IgG3/4 antibodies made by exon shuffles between the IgG3 and IgG4 wild-type genes, and families of IgG variant antibodies made by site-directed mutagenesis, were used to map the fine specificity of HRA-RFs. The fine specificity of HRA-RFs were also similar to those of EBV-RA-RFs. These studies demonstrate that the method used for immortalizing IgM, RF-producing B cells from RA patients does not influence the specificity of the RFs obtained. Furthermore, some RFs expressed in RA have distinct and unique specificities, and may therefore represent disease-specific autoantibodies.

Flow cytometry with or without cytochemistry for the diagnosis of acute leukemias?

Ninety-three (93) cases of acute leukemia were assessed using flow cytometry and cytochemistry and assigned to one of four categories: myeloid, lymphoid, biphenotypic, and non-diagnostic. In leukemias designated as ALL or AML by both methodologies, there was lineage agreement in all but 3 of 71 cases (95.8%). However, when nondiagnostic or biphenotypic diagnoses made by either methodology were included, complete agreement occurred in only 77.4% of cases. Of 37 cases designated myeloid origin by flow cytometry, 33 (89.2%) were read as myeloid by cytochemistry. The four discordant diagnosis were read as lymphoid (2) or as non-diagnostic (2). Eighty percent of lymphoid leukemias were diagnosed as such by both flow cytometry and cytochemistry; one early B cell ALL was diagnosed as myeloid and 8 as non-diagnostic. Fifty percent (50%) of flow cytometry defined T-cell ALL were considered non-diagnostic by cytochemistry as compared to 17% of the total ALL group. Of the remaining four designated non-T cell ALL by flow cytometry and non-diagnostic by cytochemistry, three were read by flow cytometry to be standard pre-B ALL and one an early B-cell ALL. Only 2/9 leukemias considered biphenotypic by flow were identified as such by cytochemistry. Given (1) the potential importance of non-lineage expression in the prognosis of myeloid and lymphoid leukemias, (2) cytochemistry's impaired ability to diagnose biphenotypic, T-cell, and promyelocytic leukemias, and (3) the increased costs incurred in diagnosis when both modalities are used, perhaps it is time to re-examine the utility of performing both flow cytometry and cytochemistry as initial testing for leukemia categorization.

Mapping IgG epitopes bound by rheumatoid factors from immunized controls identifies disease-specific rheumatoid factors produced by patients with rheumatoid arthritis.

We have mapped the specificity of 28 monoclonal IgM rheumatoid factors (RFs) produced by heterohybridomas derived from five healthy blood donors immunized with mismatched human red blood cells (HID). The HID-RFs did not differ in their binding specificity for IgG epitopes from RFs that we previously analyzed from patients with Waldenström's macroglobulinemia. However, IgM RFs produced by HID differed in their specificity for IgG compared with RFs expressed by patients with rheumatoid arthritis (RA-RFs). Only 1 of 28 HID-RFs bound all IgG subclasses (pan binding pattern) compared with 7 of 19 RA-RFs (p = 0.006). Three HID-RFs bound IgG3 compared with 9 RA-RFs (p = 0.007). Fine specificity differences were also identified between HID- and RA-RFs. Therefore, some RA-RFs show novel specificities for IgG not found among RFs from HID or individuals with Waldenström's macroglobulinemia who do not have joint disease. These Abs with unique specificities may represent disease-specific autoantibodies in patients with RA. Nine of the HID-RFs from the same individual were clonally related, and several contained somatic mutations. Even when the clonally related HID-RFs were considered as one RF for comparison, the reactivity of the HID-RFs differed significantly from RA-RFs in their inability to recognize all IgG subclasses (p = 0.044) and recognize IgG3 (p = 0.041). Interestingly, among the clonally related RFs, considerable differences in the specificity for IgG were also observed, with the RF containing the most somatic mutations in VH and VL showing the most distinctive specificity changes. Therefore, these studies also demonstrate a correlation between somatic mutation and binding specificity.

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human IgG4 Fc, at 3.2 A resolution. This is the first structure of an autoantibody-autoantigen complex. The epitope recognised in IgG Fc includes the C gamma 2/C gamma 3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

Increased frequency of HLA-DR11 in pediatric human immunodeficiency virus-associated parotid gland enlargement.

We sought to determine whether an increased frequency of the HLA-DR11 (formerly DR5) phenotype is found in human immunodeficiency virus (HIV)-infected children with parotid gland enlargement. In HIV-infected adults, parotid gland enlargement may be part of the diffuse infiltrative CD8 lymphocytosis syndrome. An increased frequency of expression of HLA-DR11 has been described in association with diffuse infiltrative CD8 lymphocytosis syndrome. We conducted a case-control study with 26 HIV-infected children, 13 of whom had parotid gland enlargement and 13 of whom did not but who were matched for age, race, and sex with those with parotid gland enlargement. Clinical and laboratory parameters (including HLA-DR11 phenotype) were compared between the two groups. HIV-positive children with parotid gland enlargement showed an increased frequency of HLA-DR11, similar to their adult counterparts with diffuse infiltrative CD8 lymphocytosis syndrome. The HLA-DR11 phenotype may be associated with the development of parotid gland enlargement in HIV-infected children and may be a marker for a more benign outcome of HIV infection.

Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants.

The neonatal Fc receptor (FcRn) binds maternal immunoglobulin G (IgG) from ingested milk in the gut (pH 6.0-6.5) and delivers it to the bloodstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reproduces the physiological pH-dependent interaction with IgG, showing high-affinity binding at pH 6.0-6.5 but weak or no binding at pH 7.0-7.5. We have studied the pH dependence of the FcRn/IgG interaction using a surface plasmon resonance assay to measure kinetic and equilibrium constants. We show that the affinity of FcRn for IgG is reduced about 2 orders of magnitude as the pH is raised from 6.0 to 7.0. A hill put analysis suggests that several titrating residues participate in the pH-dependent affinity transition. Histidine side chains are likely candidate for residues that titrate between pH 6.0 and 7.0, and previous biochemical and structural work identified several histidines on the Fc portion of IgG that are located at the FcRn binding site. Using mutant IgG molecules and IgG subtype variants that differ in the number of histidines at the IgG/FcRn interface, we demonstrate that IgG histidines located at the junction between the CH2 and CH3 domains (residues 310 and 433) contribute to the pH-dependent affinity transition. Experiments with a mutant FcRn molecule show that two histidines on the FcRn heavy chain (residues 250 and 251) also contribute to the pH dependence of the FcRn/IgG interaction. There results are interpreted using the crystal structures of FcRn and an FcRn/Fc complex.

Hyper IgM syndrome associated with defective CD40-mediated B cell activation.

Recent studies show that most patients with X-linked hyper IgM syndrome have defects in the gene for CD40 ligand. We evaluated 17 unrelated males suspected of having X-linked hyper IgM syndrome. Activated T cells from 13 of the 17 patients failed to bind a soluble CD40 construct. In these patients, the sequence of CD40 ligand demonstrated mutations. By contrast, T cells from the remaining four patients exhibited normal binding to the CD40 construct. Sequencing of the cDNA for CD40 ligand from these patients did not show mutations. The possibility that hyper IgM syndrome in these four patients was due to abnormalities in the B cell response to CD40-mediated signals was examined. Peripheral blood lymphocytes were stimulated with anti-CD40 alone, IL4 alone or anti-CD40 plus IL4. In comparison with B cells from controls or patients with hyper IgM syndrome and mutant CD40 ligand, B cells from the patients with hyper IgM syndrome and normal CD40 ligand were defective in their ability to secrete IgE (P < 0.02) or express activation markers, CD25 and CD23 (P < 0.02) in response to stimulation with anti-CD40. The failure of these B cells to respond to CD40-mediated activation could not be attributed to a generalized deficiency in B cell activation because IL4 induced normal up-regulation of CD23 and CD25 expression. These findings indicate that hyper IgM syndrome may result from defects in expression of CD40 ligand by activated T cells or defects in CD40-mediated signal transduction in B cells.

Flow cytometric analysis of natural killer cell function as a clinical assay.

The 51Cr release assay has been the method of choice in analyzing natural killer cell (NK) function. Previous FCM cytotoxicity assays of NK activity have had numerous disadvantages that discouraged clinicians from attempting to evaluate NK function by flow cytometry. We demonstrate the effectiveness of using PKH-26, a stable membrane dye, to label the K562 target cells and propidium iodide intercalation into killed target cell DNA to determine the percentage of target cells killed by effector NK cells from the peripheral blood or bone marrow. This method compares favorably with the 51Cr release assay and is quicker and easier to perform. The percentage of cytotoxicity of NK cells (CD3- CD56+ and/or CD16+) from 10 normal subjects and 10 HIV-infected children are reported to demonstrate the feasibility of studying NK function in clinical populations by FCM. The potentiation of cytolysis by alpha-interferon and interleukin 2 in vitro was also compared between these two study groups. In addition, a patient whose leukemic blasts expressed CD56+ was also studied for NK activity using this flow cytometric assay. The benefits of using this flow cytometric approach to clinically assess NK function are discussed.