A phase 2, randomized, double-blind, placebo- controlled study of chemo-immunotherapy combination using motolimod with pegylated liposomal doxorubicin in recurrent or persistent ovarian cancer: a Gynecologic Oncology Group partners study.

BACKGROUND: A phase 2, randomized, placebo-controlled trial was conducted in women with recurrent epithelial ovarian carcinoma to evaluate the efficacy and safety of motolimod-a Toll-like receptor 8 (TLR8) agonist that stimulates robust innate immune responses-combined with pegylated liposomal doxorubicin (PLD), a chemotherapeutic that induces immunogenic cell death. PATIENTS AND METHODS: Women with ovarian, fallopian tube, or primary peritoneal carcinoma were randomized 1 : 1 to receive PLD in combination with blinded motolimod or placebo. Randomization was stratified by platinum-free interval (≤6 versus >6-12 months) and Gynecologic Oncology Group (GOG) performance status (0 versus 1). Treatment cycles were repeated every 28 days until disease progression. RESULTS: The addition of motolimod to PLD did not significantly improve overall survival (OS; log rank one-sided P = 0.923, HR = 1.22) or progression-free survival (PFS; log rank one-sided P = 0.943, HR = 1.21). The combination was well tolerated, with no synergistic or unexpected serious toxicity. Most patients experienced adverse events of fatigue, anemia, nausea, decreased white blood cells, and constipation. In pre-specified subgroup analyses, motolimod-treated patients who experienced injection site reactions (ISR) had a lower risk of death compared with those who did not experience ISR. Additionally, pre-treatment in vitro responses of immune biomarkers to TLR8 stimulation predicted OS outcomes in patients receiving motolimod on study. Immune score (tumor infiltrating lymphocytes; TIL), TLR8 single-nucleotide polymorphisms, mutational status in BRCA and other DNA repair genes, and autoantibody biomarkers did not correlate with OS or PFS. CONCLUSIONS: The addition of motolimod to PLD did not improve clinical outcomes compared with placebo. However, subset analyses identified statistically significant differences in the OS of motolimod-treated patients on the basis of ISR and in vitro immune responses. Collectively, these data may provide important clues for identifying patients for treatment with immunomodulatory agents in novel combinations and/or delivery approaches. TRIAL REGISTRATION: Clinicaltrials.gov, NCT 01666444.

Complete amino acid sequences of variable regions of two human IgM rheumatoid factors, BOR and KAS of the Wa idiotypic family, reveal restricted use of heavy and light chain variable and joining region gene segments.

Evidence derived from the complete amino acid sequences of the variable regions of both the heavy and light chains of two members (BOR and KAS) of the Wa idiotypic family of human rheumatoid factors suggests that not only are the light chains of these molecules derived from possibly one variable region gene segment, but the heavy chain variable regions are all derived from the VHI subgroup of human V region genes. These molecules exhibit a surprising conservation in the size of D region, and all use the JH4 gene element. This restriction in use of VL, VH, D, and JH suggests all of these elements may play a crucial role in either antigen binding and/or expression of the crossreactive idiotype.

Molecular characterization of human Ro/SS-A antigen. Amino terminal sequence of the protein moiety of human Ro/SS-A antigen and immunological activity of a corresponding synthetic peptide.

The Ro/SS-A antigen was purified from an Epstein-Barr virus-transformed human B lymphoblastoid cell line. The amino terminal amino acid sequence of the 60-kD polypeptide bearing this antigenic epitope was determined to be: (formula; see text) A peptide composed of residue 6-19 was synthesized by the solid-phase method. Immunodiffusion-defined monospecific autoimmune sera to Ro/SS-A reacted with this synthetic peptide in ELISA, whereas autoantibodies with other specificities such as anti-La/SS-B and anti-Sm, as well as normal human sera, were not reactive. In addition, rabbit anti-peptide 6-19 antisera reacted specifically with native human Ro/SS-A antigen in ELISA. Furthermore, this synthetic peptide inhibited the binding of rabbit anti-peptide antiserum to native human Ro/SS-A. An additional synthetic peptide corresponding to residues 7-24 partially inhibited the binding of a patient anti-Ro/SS-A serum to native Ro/SS-A. These results suggest that the amino terminal portion of the molecule represents a major epitope of Ro/SS-A. The determination of the amino acid sequence of Ro/SS-A and the availability of synthetic peptide(s) bearing this antigen should provide additional approaches to further characterize the autoimmune response to this antigen.

Complete protein sequences of the variable regions of the cloned heavy and light chains of a human anti-cytomegalovirus antibody reveal a striking similarity to human monoclonal rheumatoid factors of the Wa idiotypic family.

The complete amino acid and nucleotide sequences of the variable regions of the heavy and light polypeptide chains of a human neutralizing IgGl anti-cytomegalovirus (CMV) antibody reveal a striking homology to IgM rheumatoid factors (RFs) of the Wa idiotypic family. The anti-CMV antibody and Wa RFs have in common VKIIIb, JKl, and VHIa gene segments but use different DH and JH gene segments. The anti-CMV antibody does not have RF activity and does not express the Wa idiotype. The Wa RFs do not have anti-CMV activity. A subset of Wa RFs, however, and the anti-CMV antibody do share several idiotypes on the VHIa and VKIIIb polypeptides. Since there are major differences in the antigen binding characteristics and some of the other expressed idiotypes, these data suggest that the D and J region amino acids are crucial to such specificities. Although the use of such highly homologous gene segments in different immune responses is well-documented in murine systems, these data represent the first such example in the human.

Molecular cloning, expression, and chromosome 19 localization of a human Ro/SS-A autoantigen.

Ro/SS-A antibodies are found in a number of human autoimmune disorders including Sjogren's syndrome and several systemic lupus erythematosus-related disorders. These heterogeneous autoantibodies are known to recognize several distinct cellular antigens. With synthetic oligonucleotides corresponding to amino acid sequence information we have isolated a full-length cDNA clone which encodes a human Ro ribonucleoprotein autoantigen. The 1,890-base pair clone contains an open reading frame that encodes a 417-amino acid, 48-kD polypeptide that migrates aberrantly at 60 kD by SDS-PAGE. Rabbit antibodies raised against this protein's recently described amino-terminal epitope react with a previously identified 52-kD human Ro protein and immunoprecipitate the human cytoplasmic RNAs. Ultraviolet light cross-linking studies suggest that this Ro protein binds each of the four major human cytoplasmic RNAs. The deduced amino acid sequence is 63% homologous to an Onchocerca volvulus antigen. Southern filter hybridization analysis indicates that this gene is not highly polymorphic and exists as a single copy in the human genome. Chromosomal localization studies place this gene on the short arm of chromosome 19 near the gene encoding the low density lipoprotein receptor.

Two microsomal-associated iron-binding proteins observed in rat small intestinal cells during iron absorption.

Acrylamide gel electrophoresis of microsomal protein obtained from rat small intestinal mucosal cells, after an injection of [3H]leucine, demonstrated increased quantities of two soluble iron-binding proteins during iron absorption, one with a high molecular weight (about 400 000) and the other of intermediate molecular weight (80 000). Both proteins were present in a ribosomal-enriched sub-fraction obtained during purification of the microsomal membrane but were not identified among the purified membrane proteins.

Anti-DNA and anti-platelet specificities of SLE-derived autoantibodies: evidence for CDR2H mutations and CDR3H motifs.

Although polyreactivity appears to be a characteristic feature of natural autoantibodies, polyreactive anti-DNA autoantibodies can be derived both from patients with autoimmune disease and from normal individuals. It is unclear whether these autoantibodies differ depending on their origin, but previous studies from our laboratory have suggested that polyreactive systemic lupus erythematosus (SLE)-derived platelet-binding anti-DNA autoantibodies have more restricted antigen reactivity and greater functional activity than normal-derived polyreactive autoantibodies. The objective of the present study was to characterize the VH and VL region sequences of 10 human hybridoma anti-DNA autoantibodies derived from peripheral blood lymphocytes of different origins [SLE, rheumatoid arthritis (RA), or normal] to determine whether there are structural differences between these autoantibodies. We show that although some unmutated germline structures (VH and VL) are represented, these are not restricted to anti-DNA autoantibodies from normal individuals and that two normal-derived anti-DNA antibodies showed quite extensively mutated VH genes. However, these mutations, unlike those found in the CDR2H of several of the SLE-derived antibodies, did not appear to be antigen-selected. Three different amino acid motifs, putatively involved in antigen binding specificity, were observed in the CDR3H segments of some of the autoantibodies. One was the previously described YYGSG motif, which was found in a normal-derived anti-DNA autoantibody, while two new potential motifs were observed only in SLE-derived platelet-binding anti-DNA autoantibodies. These data suggest that antigenic and functional differences between SLE-derived and normal-derived platelet-binding anti-DNA autoantibodies may be due to antigen-selected mutations in the CDR2H and specific amino acid motifs in the CDR3H.

Amino acid sequence of a light chain variable region of a human rheumatoid factor of the Wa idiotypic group, in part predicted by its reactivity with antipeptide antibodies.

Antipeptide antisera were raised to the second and third complementarity-determining regions of the light chain derived from a human monoclonal IgM (Sie) which had antigammaglobulin activity and belonged to the Wa cross-reactive idiotypic group of human rheumatoid factors, two of whose members (Sie, Wo1) had been previously sequenced in our laboratory (Andrews and Capra, Biochemistry 20, 5816-5822, 1981). These antisera were found to react with the light chain of another human monoclonal IgM (Go1) that shared the Wa idiotype while antipeptide antisera made to the third CDR of the Sie heavy chain failed to react. The amino acid sequence of the variable region of the Go1 light chain was found to be highly homologous to the light chain of Sie from which the synthetic peptides were derived, particularly in the framework regions and the second and third CDR. This study illustrates that antipeptide antisera are valuable and specific probes for determining the relationship between molecules which exhibit similar antigen binding or idiotypic specificities and, furthermore, such antisera are able to predict amino acid sequences with surprising precision.

Restricted variable region gene usage and possible rheumatoid factor relationship among human monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B.

The nucleotide sequences of the variable region genes encoding five different human, high affinity antibodies, specific for the major neutralization determinant (AD-1) expressed by human cytomegalovirus glycoprotein B (gp58/116), have been determined. Three of the five heavy chain variable regions belonged to the small VHV-family, although they combined with a diverse set of light chains (V kappa IIIb, V lambda II and V lambda III). The other two antibodies belonged to VH-families III and IV. One of the VHV-family genes most likely originated from a previously unreported germline gene or allele, since it carries a nine nucleotide insert in framework 1. In addition, V lambda-genes showed variable homology (77-95%) to known germline sequences, while V kappa-genes showed high homology (approximately 98%) with their proposed germline origin. Despite the close homology of the V kappa IIIb-gene used to express one of the antibodies with its corresponding germline gene, the protein did not strongly express some idiotypes associated with this light chain family. There is, thus, no direct relation between the expression of these crossreactive idiotypes and the use of even modestly mutated light chains belonging to this V kappa-family, which has been implicated in the development of anti-idiotypic networks possibly inducing autoantibodies, such as rheumatoid factors.

Structural characteristics of four human hybridoma antibodies specific for the pp65 protein of the human cytomegalovirus and their relationship to human rheumatoid factors.

Four human hybridoma antibodies directed against the human cytomegalovirus (CMV) were characterized with respect to their immunoglobulin gene usage and expression of rheumatoid factor (RF) associated idiotypes and variable region epitopes. The aims of these experiments were: (1) to characterize the immunoglobulin gene usage of four antibodies directed against a single protein of a human pathogen; and (2) to examine how this humoral response may be linked to the production of RFs, autoantibodies found in the majority of patients with rheumatoid arthritis (RA). All four anti-CMV antibodies were of the gamma heavy chain isotype and were specific for the immunodominant 65 kDa viral matrix phosphoprotein (pp65). The four anti-pp65 antibodies expressed different light (L) and heavy (H) chain variable region gene combinations. These were: VkIII/VH3, V lambda 1/VH3, V lambda 1/VH4 and V lambda 3/VH3, respectively for the HCV-2, HCV-3, HCV-63 and HCV-65 hybridoma cell lines. Although none had RF activity, each of these antibodies expressed a unique set of RF-associated determinants, implying different three-dimensional configurations of the variable regions of these antibodies. The HCV-2 antibody, however, had the most extensive similarities to human RFs since it not only expressed the greatest number of RF-associated determinants but also had a protein sequence that was very homologous to RFs of the "Po" idiotypic family. Furthermore, predicted germline gene usage by anti-CMV antibodies and RFs suggest that some are encoded by identical or similar genes and that the different specificities are achieved by somatic mutations in the L and H chain complementarity determining regions (CDRs) and genetic diversity in the H chain CDR3.

Biochemical properties of a novel rabbit thymocyte specific class I-like antigen.

Monoclonal antibody 5E2 identifies a new rabbit thymocyte specific cell surface molecule designated R-Ta. SDS-PAGE of molecules immunoprecipitated by 5E2 shows that R-Ta exists as a non-covalently associated hetero-dimer consisting of a light polypeptide chain (mol. wt approximately 12,000) and a bi-molecular species of a heavy chain (mol. wts of 45,000 and 40,000). The difference between the two forms of heavy chain can be attributed to different degrees of glycosylation. Each form of the R-Ta heavy chain has a polypeptide mol. wt of 34,000. At least three N-linked oligosaccharides and no significant O-linked sugars were found associated with R-Ta. Two dimensional electrophoresis of V8 protease peptide maps also indicate that the two forms of the heavy chains are similar, if not identical, in polypeptide primary structure. The light polypeptide was found to be serologically and structurally identical to beta-2-microglobulin. This was demonstrated in a previous study by reaction with goat anti-beta-2-microglobulin antisera. In this investigation the structural identity with beta-2-microglobulin was demonstrated by partial amino terminal sequence analysis. The partial amino acid sequence for 18 steps of the R-Ta heavy chain was also determined. A comparison of the amino acid sequence with other known sequences for the conventional Class I molecules of man, mouse and rabbit did not reveal any homology. Thus R-Ta is a new T-cell surface protein, and like human CD1, carries the unique distinction of thymocyte specificity, is beta-2-microglobulin associated, but is not Class I related.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis.

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Identification of IgG rheumatoid factors by a novel method utilizing immunoblotting.

Monoclonal IgG rheumatoid factors (RFs) are identified using a novel immunoblot assay which detects RFs that bind to SDS-denatured Fc on nitrocellulose. Recognition of these self-associating antibodies is by the use of F(ab')2 fragments of light chain-specific antisera. In this way, IgG RFs can be easily identified and the precise binding characteristics to different isotypes of IgG, or other antigens, further specified. The assay can also be used to detect other classes of RFs such as IgM RFs. Although less sensitive than the standard ELISA, the use of this immunoblot RF assay (IRFA) will identify IgG RFs and their target antigen with precision.

Aberrant cellular localization of rubella viral genome in patients with adult Still's disease--a pilot study.

The rubella virus (RV) genome was detected using polymerase chain amplification techniques in several peripheral blood cell populations in patients with adult Still's disease (ASD) and normal controls (NC), including mononuclear cells (PBMC), B-cells, T-cells, monocyte/macrophages, and polymorphonuclear leukocytes (PMN). Five of 6 ASD patients and 3 of 6 NC subjects had detectable RV genome. Viral genomic load was significantly higher in ASD than in NC subjects (4.4 fold higher, p = 0.03). Interestingly, a differential cellular distribution of viral genome was observed between ASD and NC individuals. RV genome was detected more frequently in the PBMCs of ASD (5 of 6) patients compared to 2 of 6 NC. The viral genome was more localized to the PMN compartment equally in ASD and in NC subjects. On further cellular analysis, RV genome was detected in B-cells and macrophages but not T-cells in one patient. Existence of a differential viral genomic reservoir between ASD and NC suggests that this may play a role in the pathogenesis of disease manifestations and may reflect the inability to clear latent virus.

Perifoveal vitreous detachment is the primary pathogenic event in idiopathic macular hole formation.

OBJECTIVE: To evaluate the relationship between the posterior vitreous cortex and the posterior retina in eyes with early stages of idiopathic macular hole formation. METHODS: Twenty-six eyes of 26 consecutive patients with stage 1 or stage 2 idiopathic macular hole underwent complete ophthalmologic examination, contact lens biomicroscopy, and B-scan ultrasonography or vitreoretinal surgery or both. In eyes that were operated on, the posterior cortical vitreous layer was meticulously examined with a silicone-tipped cannula prior to inducing a posterior vitreous detachment. RESULTS: In 25 (96%) of 26 eyes, one or more examination techniques revealed a shallow, localized detachment of the perifoveal vitreous, typically extending to the level of the vascular arcades. Among these 25 eyes, the posterior hyaloid membrane separation was detectable biomicroscopically in 4 (16%) of 25 eyes, ultrasonographically in 17 (74%) of 23 eyes, and intraoperatively in 23 (100%) of 23 eyes. Persistent vitreous adherence to the foveola was evident in 6 (100%) of 6 eyes with a stage 1 hole and in 12 (92%) of 13 eyes with a stage 2 hole but no operculum. CONCLUSIONS: These findings suggest that localized perifoveal vitreous detachment (an early stage of age-related posterior vitreous detachment) is the primary pathogenic event in idiopathic macular hole formation. We postulate that detachment of the posterior hyaloid from the pericentral retina leads to foveal dehiscence by exerting anterior traction on the foveola and by localizing into the foveola the dynamic vitreous traction associated with ocular rotations.

Removal of hepatitis B surface antigen from a contaminated applanation tonometer.

Hepatitis B surface antigen was found in the conjunctival fluid of 43% of hepatitis B surface antigen carriers and could be detected on the tonometer tip after tonometry in one of four hepatitis B surface antigen carriers whose conjunctival fluid was positive for hepatitis B surface antigen. The Goldmann applanation tonometer tip was contaminated by immersion into solutions containing high concentrations of hepatitis B surface antigen (average 15,000 cpm). Prompt rinsing of the contaminated tonometer in running tap water for ten seconds proved sufficient to remove all detectable hepatitis B surface antigen from the tonometer.

Follow-up and ultrasonographic examination of patients with macular pseudo-operculum.

Twenty-nine patients (30 eyes) with pseudo-opercula were followed up for six to 65 months (mean, 24 months). Visual acuity was 20/30 or better in all but one eye that was amblyopic. Only one eye developed a decrease in visual acuity and changes interpreted as a stage 1 (foveal detachment induced by tangential traction of the cortical vitreous) impending hole. After vitrectomy visual acuity returned to 20/20. Ultrasonographic examination confirmed the presence of pseudo-opercula in all 22 eyes examined. We concluded that the a pseudo-operculum is a favorable prognostic sign and that its presence is demonstrable ultrasonographically.

Methods for the Hong Kong Vision Study: a pilot assessment of visual impairment in adults.

INTRODUCTION: The Hong Kong Vision Study (HKVS) was a pilot study to collect data on the prevalence of eye diseases and risk factors in Hong Kong using methodology comparable to that developed in America and Australia. AIM: The main goal was: to evaluate the application of the methodology in a different culture and language; and to determine the prevalence and risk factors of eye diseases in order to design a larger study of an ethnic Chinese population. METHOD: This study was patterned after the Melbourne Visual Impairment Project using the Chinese language in data collection and examinations. CONCLUSION: Well-designed methodology is transferable to different cultures, languages and continents. Use of similar methodology will enable better comparisons and analyses to be made from population-based data.

A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized.

A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.

The Hong Kong vision study: a pilot assessment of visual impairment in adults.

PURPOSE: The Hong Kong Adult Vision Pilot Study is a population based study of the distribution and determinants of eye disease in a random sample of the Chinese population age 40 and over. The present pilot study identifies the extent and causes of visual loss using methods developed in the United States and Australia. The pilot study uses the prevalence data to estimate the sample size necessary to predict the size of an effect a larger study may detect and the confidence with which that effect may be considered and the standard deviation of the Hong Kong population. The smallest detectable odds ratios were calculated based on known risk factor prevalence rates of the pilot study. METHODS: Hong Kong Chinese residents aged 40 and over in 2 random cluster sites were identified by private household census. The examinations were performed at one location and included, health history and habits, presenting and best corrected LogMar vision, Humphrey visual field and IOP measurement, dilated slit lamp, fundus examination, fundus photography and echography. RESULTS: In the two test sites, 355 people were examined of the 441 eligible residents (81% response). 76.6% of the population reported a change in vision in the last 10 years; 45% had not sought examination. 4.54% had vision less than 20/60. This was caused by: myopic choroidal degeneration (31%), cataract (19%), cataract + ARM (19%), ARMD (19%), glaucoma (6%), and corneal disease (6%). Vision loss increased significantly with age. Vision loss was more common in older women than in older men. The prevalence rates calculated from the pilot study data were used, requiring a relative precision of 95% and +/- 20% confidence interval of the prevalence rates, indicate that a sample size of 2500 would be a good number for a larger study. CONCLUSIONS: The methods developed in the United States and Australia for completing eye disease prevalence studies are applicable in Hong Kong. Vision loss is increasingly common in older people and the percent of visual impairment in Hong Kong is higher than studies in the US and Australia. As the population ages demands on the health care systems will increase. The results from this pilot warrant continuation of the study. Efforts must be directed toward prevention of visual loss.