The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities - that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.

A method to quantitate myeloperoxidase (MPO) activity from rat whole kidney is described. Polymorphonuclear leukocyte (PMN) infiltration into tissue is a hallmark of acute inflammation. Historically, the degree of inflammation has been quantified by the identification and enumeration of PMNs histologically or by some other means. More recently, the enzyme activity of MPO, a marker enzyme for PMN, and freshly emigrated monocytes in many inflamed tissues has replaced these methods. The kidney, however, has been identified as a tissue from which MPO cannot be measured. Indeed, kidney homogenized by a standard extraction procedure was devoid of MPO activity. We modified the established methodology so that kidney was homogenized in 5 mM potassium phosphate buffer (PB) first and then centrifuged at 30,000 g for 30 min at 4 degrees C prior to extraction. The resulting 30,000 g pellets expressed MPO activity after suspending them in 50 mM PB containing 0.5% hexadecyltrimethylammoniumbromide (HTAB). Interference in the assay was observed with supernatants from control and inflamed kidney, which appeared to be due to kidney-derived material forming a complex with HTAB. After washing the pellets twice, we noted that their extracts exhibited greater activity, and interference from supernatants was abolished. Using this method, we observed that acutely inflamed kidneys from rats treated with sheep nephrotoxic immunoglobulin G (IgG) had significantly elevated MPO activity over kidneys from control rats. Thus, the described technique allows for the routine assay of MPO in kidney tissue.

The neonatal Fc receptor (FcRn) performs two distinct but related functions: transport of maternal immunoglobulin G (IgG) to pre- or neonatal mammals, thus providing passive immunity, and protection of IgG from normal serum protein catabolism. FcRn is related to class I MHC proteins but lacks a functional peptide binding groove. The crystal structure of human FcRn has been determined at 2.7 A resolution and compared to the previously described structure of rat FcRn [Burmeister et al. (1994) Nature 372, 336-343] and to the structures of MHC and MHC-related proteins. Human FcRn is structurally similar to the rat receptor but does not form receptor dimers in the crystals as observed in crystals of rat FcRn. The interaction between human FcRn and IgG was characterized by determining the binding stoichiometry using equilibrium gel filtration and by deriving binding affinities for the different human IgG subclasses using a surface plasmon resonance assay. Like rat and mouse FcRn, human FcRn interacts with IgG with a 2:1 receptor:ligand stoichiometry. The binding of human FcRn to the four human IgG subclasses shows subclass and allotype variations but no clear subclass affinity differences that correlate with serum half-lives. The structure of human FcRn and studies of its ligand binding are relevant to current efforts to use FcRn-mediated regulation of IgG half-life in serum to increase the lifetimes of antibody-based therapeutics.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase eta, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase iota gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of pol eta-deficient mice differs markedly. This suggests that, in the absence of pol eta, the MSH2-MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly pol kappa, to assume its function in hypermutation.

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.

The programmed development of lymph nodes and Peyer's patches during ontogeny requires lymphoid tissue inducer (LTi) cells that express the nuclear hormone receptor RORγt. After birth, LTi cells in the intestine cluster into cryptopatches, the precursors of isolated lymphoid follicles (ILFs), which are induced to form by symbiotic bacteria and maintain intestinal homeostasis. We show that in RORγt-deficient mice, which lack LTi cells, programmed lymphoid tissues, ILFs, and Th17 cells, bacterial containment requires the generation of large numbers of tertiary lymphoid tissues (tLTs) through the activity of B cells. However, upon epithelial damage, these mice develop severe intestinal inflammation characterized by extensive recruitment of neutrophils and IgG(+) B cells, high expression of activation-induced deaminase in tLTs, and wasting disease. The pathology was prevented by antibiotic treatment or inhibition of lymphoid tissue formation and was significantly decreased by treatment with intravenous immunoglobulin G (IVIG). Our data show that intestinal immunodeficiency, such as an absence in RORγt-mediated proinflammatory immunity, can be compensated by increased lymphoid tissue genesis. However, this comes at a high cost for the host and can lead to a deregulated B cell response and aggravated inflammatory pathology.

We have studied the antibody responses to Env and Gag antigens of human immunodeficiency virus type 1 (HIV-1) in several cohorts of HIV-1-infected individuals: long-term nonprogressors, progressors to disease, acute seroconvertors, and recipients of HIV-1 protease inhibitors. We conclude that the antibody responses to Env and Gag antigens are differentially regulated and that changes in the plasma viral load in the measurable range (500 to 10(8) RNA copies per ml) do not directly affect the antibody responses to these HIV-1 proteins. We provide quantitative estimates of HIV-1-specific immunoglobulin G concentrations in plasma, which can be in excess of 1 mg/ml for both anti-gp120 and anti-p24 once the immune response to HIV-1 has stabilized after seroconversion. We discuss the apparent paradox that the absence of anti-Gag antibodies (which have, at best, limited antiviral activity) is indicative of disease progression, while the retention of anti-Env antibodies (which do have antiviral activity) is of limited (or no) prognostic value. We show that the disappearance of anti-Gag antibodies during disease progression is highly unlikely to be due to immune complexing; instead, we believe that it reflects the loss of T-cell help that is more necessary for the anti-Gag than the anti-Env response.

OBJECTIVE

The diagnosis of drug-induced liver injury relies on exclusion of other causes, including viral hepatitis A, B, and C. Hepatitis E virus (HEV) infection has been proposed as another cause of suspected drug-induced liver disease. We assessed the frequency of HEV infection among patients with drug-induced liver injury in the United States.

METHODS

The Drug-Induced Liver Injury Network (DILIN) is a prospective study of patients with suspected drug-induced liver injury; clinical information and biological samples are collected to investigate pathogenesis and disease progression. We analyzed serum samples, collected from patients enrolled in DILIN, for immunoglobulin (Ig) G and IgM against HEV; selected samples were tested for HEV RNA.

RESULTS

Among 318 patients with suspected drug-induced liver injury, 50 (16%) tested positive for anti-HEV IgG and 9 (3%) for anti-HEV IgM. The samples that contained anti-HEV IgM (collected 2 to 24 weeks after onset of symptoms) included 4 that tested positive for HEV RNA genotype 3. Samples from the 6-month follow-up visit were available from 4 patients; they were negative for anti-HEV IgM, but levels of anti-HEV IgG increased with time. Patients who had anti-HEV IgM were mostly older men (89%; mean age, 67 years), and 2 were human immunodeficiency virus positive. Clinical reassessment of the 9 patients with anti-HEV IgM indicated that acute hepatitis E was the most likely diagnosis for 7 and might be the primary diagnosis for 2.

CONCLUSIONS

HEV infection contributes to a small but important proportion of cases of acute liver injury that are suspected to be drug induced. Serologic testing for HEV infection should be performed, particularly if clinical features are compatible with acute viral hepatitis.

The expression of HLA-G at the fetal-maternal interface during pregnancy and in transplanted tissue makes this a key molecule in the acceptance of a semiallogeneic fetus and allogeneic transplant. Dendritic cells (DC) play a critical role in the control of innate and adaptive immune responses. DC are present in maternal decidua, but must be kept under tight control. Here we describe the mechanism of tolerization of DC by HLA-G through inhibitory receptor interactions. The HLA-G-ILT (immunoglobulin-like transcript) interaction leads to development of tolerogenic DC with the induction of anergic and immunosuppressive T cells. Using human monocyte-derived DC and ILT4-transgenic mice, we show that (i) HLA-G induces the development of tolerogenic DC with arrest maturation/activation of myeloid DC, (ii) HLA-G-modified DC induce differentiation of anergic and immunosuppressive CD4(+) and CD8(+) effector T cells, and (iii) the gene expression profile provides evidence that HLA-G induces tolerogenic DC by disruption of the MHC class II presentation pathway. Ligation of ILT4 receptor on DC from transgenic mice diminished peptide presentation by MHC class II molecules and significantly prolonged allograft survival. These findings provide support that HLA-G is an important tolerogenic molecule on DC for the acceptance of a semiallogeneic fetus and transplanted tissue/organ.

Contact between human neutrophils and aggregated immunoglobulin G bound to micropore filters has been studied as a model of the pathogenesis of tissue damage in immune complex disease. Contact with this surface, as well as with plain filters and polystyrene petri dishes, induced neutrophils to elaborate superoxide anion and hydrogen peroxide and to generate chemiluminescence, which has been attributed to singlet oxygen. Pretreatment of the cells with cytochalasin B decreased these activities but increased release of lysosomal beta-glucuronidase, suggesting that degranulation and the burst of oxygen metabolism that characterizes phagocytes are independently regulated functions. Toxic oxygen metabolites released from neutrophils are highly reactive and could mediate tissue injury at sites of inflammation.

The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh alpha [gene symbol PCDHA], Pcdh beta [PCDHB], and Pcdh gamma [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large "variable" region exon, while the intracellular domains are encoded by three small "constant" region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdh alpha and Pcdh gamma gene clusters in the two species is virtually identical, whereas the mouse Pcdh beta gene cluster is larger and contains more genes than the human Pcdh beta gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.

Cloning and nucleotide sequence determination suggest that the rearranged gamma 1-chain gene in a gamma 1-chain-producing myeloma a-pears to be formed by the recombination between the 5' flanking regions of the gamma 1- and mu-chain genes of undifferentiated cells. The recombination site is distinct from the putative J region and is a novel region that we call the S region. We have extended our previous model that explains the heavy-chain class switching by two or more successive recombination events.

Monoclonal antibodies were prepared by the fusion of murine myeloma NS1 cells with spleen cells of BALB/c mice immunized with Formalin-killed elementary bodies of the Chlamydia trachomatis L2 serovar. The specificity of these monoclonal antibodies was determined with a solid-phase immunoassay in which HeLa 229 cells infected with C. trachomatis serovars D, <em>G</em>, H, I, L2 and the Chlamydia psittaci meningopneumonitis strain Cal-10 were used. An <em>immunoglobulin</em> <em>G</em>3 monoclonal antibody (L2I-6) was identified that reacted with both C. trachomatis- and C. psittaci-infected HeLa cells. The immunoreactivity of the genus-specific epitope was heat resistant (100 degrees C, 10 min) but was destroyed by sodium metaperiodate treatment. Further characterization of the chlamydial specificity of monoclonal antibody L2I-6 by microimmunofluorescence showed that it was reactive with all 15 C. trachomatis serovars and seven C. psittaci strains isolated from five different animal species. We undertook studies to identify the biochemical nature of the chlamydial component on which the genus-specific epitope was located. The immunoreactive component was isolated by hot phenol-water extraction of dithiothreitol-reduced chlamydial elementary bodies. The component was positive in the Limulus amoebocyte lysate test (results of Limulus amoebocyte lysate assay were identical with those of Salmonella typhimurium LT2 SAI 377 Re lipopolysaccharide [LPS]), contained 8.8% 2-keto-3-deoxyoctulosonic acid, was resistant to proteinase K, and possessed electrophoretic mobility and silver-staining characteristics in sodium dodecyl sulfate-polyacrylamide gel electrophoresis consistent with a rough LPS or glycolipid. On the basis of these findings, we conclude that the genus-specific epitope recognized by monoclonal L2I-6 is located on chlamydial LPS. We further characterized the antigenic properties of the chlamydial LPS epitope by examining the immunoreactivity of monoclonal antibody L2I-6 by immunoblotting analyses against isolated LPSs extracted from Neisseria gonorrhoeae, S. typhimurium, and Escherichia coli. Monoclonal antibody L2I-6 did not bind LPS of these organisms, demonstrating that the chlamydial genus-specific LPS epitope is apparently not shared by these gram-negative bacteria. We were able, however, to show that the chlamydial LPS does share antigenic determinants with LPS of gram-negative organisms. Polyclonal rabbit antisera raised against S. typhimurium Re LPS or lipid A showed intense immunological cross-reactivity with chlamydial LPS by immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)

We assessed the relationship of fecal environmental contamination and environmental enteropathy. We compared markers of environmental enteropathy, parasite burden, and growth in 119 Bangladeshi children (≤ 48 months of age) across rural Bangladesh living in different levels of household environmental cleanliness defined by objective indicators of water quality and sanitary and hand-washing infrastructure. Adjusted for potential confounding characteristics, children from clean households had 0.54 SDs (95% confidence interval [CI] = 0.06, 1.01) higher height-for-age z scores (HAZs), 0.32 SDs (95% CI = -0.72, 0.08) lower lactulose:mannitol (L:M) ratios in urine, and 0.24 SDs (95% CI = -0.63, 0.16) lower immunoglobulin G endotoxin core antibody (IgG EndoCAb) titers than children from contaminated households. After adjusting for age and sex, a 1-unit increase in the ln L:M was associated with a 0.33 SDs decrease in HAZ (95% CI = -0.62, -0.05). These results are consistent with the hypothesis that environmental contamination causes growth faltering mediated through environmental enteropathy.

BACKGROUND

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by antibody deficiency and a decrease in serum IgG and IgA, IgM, or both levels at least 2 SDs below the mean for age and not attributed to other known immunologic disorders. These patients often present with frequent and severe episodes of pneumonia before diagnosis. The standard treatment, intravenous immunoglobulin (IVIG), has been available for the past 20 years. No large-scale study has compared the incidence of pneumonia in these patients before and after IVIG treatment.

OBJECTIVE

The aim of this study was to document the effectiveness of intravenous immunoglobulin treatment on the incidence of pneumonia in patients with CVID.

METHODS

We performed chart reviews and interviews of patients with laboratory-confirmed CVID seen at our clinical center. The number of episodes of pneumonia was documented before and after treatment with immunoglobulin replacement therapy.

RESULTS

The histories of 50 patients were reviewed (mean current age, 42 +/- 16.3 years; age range, 10-78 years; 20 male and 30 female patients). Forty-two (84%) of the 50 patients with CVID had pneumonia at least once before receiving immunoglobulin treatment, and 11 of 42 of these patients had multiple episodes. After treatment with gamma globulin over a mean period of 6.6 +/- 5.2 years (range, <1-20 years), the number of patients experiencing pneumonia significantly decreased to 11 (22%) of 50. In most cases these patients had pneumonia in the first year of immunoglobulin treatment.

CONCLUSIONS

The treatment of CVID with IVIG significantly reduces the incidence of pneumonia.

The antibody kinetics in sera from 27 adults after three doses of the Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine was studied. The vaccinees received the third dose 4 to 5 years after the first two. Antibody responses against outer membrane proteins (OMPs) and lipopolysaccharides were studied by enzyme-linked immunosorbent assay and immunoblotting and with serum bactericidal assays (SBA) with three variants of the vaccine strain, 44/76. Six weeks after the second injection, the geometric mean (GM) of the levels of immunoglobulin G (IgG) against OMVs was about sevenfold higher than that of prevaccination levels, and 74% of the vaccinees developed a greater-than-twofold rise in SBA titer. After 6 months, the GM of IgG levels declined to about threefold higher, and after 4 to 5 years it declined to about twofold higher, than that before vaccination. The third dose induced a rapid increase in SBA titers in 96% of the vaccinees, and the GM of levels of IgG against OMVs rose to about 14-fold the prevaccination level. One year later, the IgG antibody levels had dropped to 4.6-fold the prevaccination level, but 88% of the vaccinees still showed bactericidal activity. The response after the two first doses was higher in individuals with prevaccination antibodies, but no such effect was found after three doses. The use of defined mutants in SBA and linear multiple regression analyses indicated that among the major OMPs, antibodies to the Opc and class 1 proteins made the most important single contributions to the bactericidal activity against the vaccine strain, but it also demonstrated the importance of antibodies against other antigens. After three doses, 68% of the vaccinees showed a significant SBA response against a strain lacking both the Opc and the class 1 proteins. Three doses converted almost all subjects to SBA responders and gave higher antibody levels and relatively less serosubtype-specific bactericidal activity than did two doses, probably indicating a broader cross-protection against heterologous strains.

We have examined secretory antibody and cell-mediated immune responses to oral cholera vaccine in the human gastrointestinal mucosa. Freshly isolated peripheral blood lymphocytes and intestinal lymphocytes obtained by enzymatic dispersion of duodenal biopsies were assayed for numbers of total and vaccine specific immunoglobulin-secreting cells by enzyme-linked immunospot assay (ELISPOT) techniques; the frequency of cells secreting interferon-gamma (IFN-gamma) was also examined by a new modification of the ELISPOT technique. After booster immunizations with oral cholera vaccine, large numbers of cholera toxin-specific antibody-secreting cells (ASC) appeared in the small intestine. The responses were dominated by IgA ASC. A single immunization, performed 5 mo after the initial vaccinations, gave rise to an ASC response similar to that seen after the first booster immunization, with respect to both magnitude and isotype distribution. Each of the immunizations also evoked an ASC response in blood which was of lower magnitude than that seen in the small intestine, and comprised similar proportions of IgA and IgG ASC. A booster immunization also resulted in increased frequencies of IFN-gamma-secreting cells, but this increase was confined to the duodenal mucosa. This study establishes the feasibility of studying, at the single-cell level, intestinal immune reactivity in humans. Furthermore, it indicates that the small intestinal mucosa is an enriched source of IFN-gamma. It also demonstrates marked differences between intestinal and peripheral blood immune responses after enteric immunization, and confirms the notion that the mucosal immune system in humans displays immunological memory.

The c-maf protooncogene is a T helper cell type 2 (Th2)-specific transcription factor that activates the interleukin (IL)-4 promoter in vitro. Although it has been postulated that c-maf directs the Th2-specific expression of the IL-4 gene in vivo, direct evidence that c-maf functions during the differentiation of normal, primary T cells is lacking. We now demonstrate that overexpression of c-maf in vivo skews the Th immune response along a Th2 pathway, as evidenced by increased production of Th2 cytokines and the IL-4-dependent immunoglobulins, IgG1 and IgE. The overproduction of IgGl and IgE in the CD4 promoter/c-maf transgenic mice was IL-4 dependent since this was not observed in c-maf transgenic mice bred onto an IL-4-deficient background. Ectopic expression of c-maf in mature Th1 cells did not confer on them the ability to produce IL-4, but did decrease the production of IFN-gamma. The attenuation of Th1 differentiation by c-maf overexpression occurred by a mechanism that was independent of IL-4 and other Th2 cytokines, and could be overcome by IL-12. These studies demonstrate that c-maf promotes Th2 differentiation by IL-4-dependent mechanisms and attenuates Th1 differentiation by Th2 cytokine-independent mechanisms.

Immunoglobulins (Igs) are found thus far only to be produced by differentiated B lymphocytes. By immunohistochemistry analysis, in situ hybridization, and laser capture microdissection-assisted single-cell PCR, we demonstrate that human cancers of epithelial origin, including carcinomas of breast, colon, liver, lung, established epithelial cancer lines, as well as some normal lung tissues, also produce IgG in both cytoplasmic and secreted forms. Furthermore, blockade of tumor-derived IgG by either antisense DNA or antihuman IgG antibody increased programmed cell death and inhibited growth of cancer cells in vitro. More importantly, administration of antihuman IgG antibody also suppressed the growth of an IgG-secreting carcinoma line in immunodeficient nude mice. Our results support a role of tumor-derived IgG as growth factor for epithelial cancers. Prevalent expression of IgG in human carcinomas and its growth-promoting functions may have important implications in growth regulation and targeted therapy of human cancers.

Neutralizing antibodies are necessary and sufficient for protection against infection with vesicular stomatitis virus (VSV). The in vitro neutralization capacities and in vivo protective capacities of a panel of immunoglobulin G monoclonal antibodies to the glycoprotein of VSV were evaluated. In vitro, neutralizing activity correlated with avidity and with neutralization rate constant, a measure of on-rate. However, in vivo, protection was independent of immunoglobulin subclass, avidity, neutralization rate constant, and in vitro neutralizing activity; above a minimal avidity threshold, protection depended simply on a minimum serum concentration. These two biologically defined thresholds of antibody specificity offer hope for the development of adoptive therapy with neutralizing antibodies.

Cytomegalovirus (CMV), a prevalent pathogen, causes severe disease in immunocompromised humans. However, present understanding is limited regarding the long-term clinical effect of persistent CMV infection in immunocompetent adults. The authors conducted a prospective observational cohort study (1992-2002) of 635 community-dwelling women in Baltimore, Maryland, aged 70-79 years in the Women's Health and Aging Studies to examine the effect of CMV infection on the risk of frailty, a common geriatric syndrome, and mortality in older women. The effect of baseline serum CMV antibody (immunoglobulin G) concentration on the risk of 3-year incident frailty, defined by using a 5-component measure, and 5-year mortality was examined with Cox proportional hazards models. Compared with those who were CMV seronegative, women in the highest quartile of CMV antibody concentration had a greater incidence of frailty (hazard ratio = 3.46, 95% confidence interval: 1.45, 8.27) and mortality (hazard ratio = 3.81, 95% confidence interval: 1.64, 8.83). After adjustment for potential confounders, CMV antibody concentration in the highest quartile independently increased the risk of 5-year mortality (hazard ratio = 2.79, 95% confidence interval: 1.22, 6.40). Better understanding of the long-term clinical consequences of CMV infection in immunocompetent humans is needed to guide public health efforts for this widely prevalent infection.

In order to understand the pharmacology of monoclonal antibodies and their conjugates, one must consider both global and microscopic aspects of antibody distribution. Here we present an analysis of antibody distribution in tumors based on the following factors: (a) molecular weight and valence of the antibody; (b) global pharmacokinetic profile following i.v. bolus injection; (c) penetration through the vascular wall; (d) diffusive and convective transport through interstitial space in the tumor; (e) antigen-antibody interaction; (f) antibody metabolism. Partial differential equations were developed to incorporate these factors and then solved numerically using parameter values from animal experiments, from clinical protocols at our institution, from studies of antibody binding characteristics in vitro, and from the literature. Salient findings from this model are that (a) antigen-antibody interaction in the tumor can retard antibody percolation away from blood capillaries, thus constituting a "binding site barrier"; (b) high antibody affinity tends to decrease antibody penetration and result in a more heterogeneous distribution; (c) high molecular weight [IgG greater than F(ab')2 greater than Fab] slows percolation and results in less uniform spatial distribution; (d) the average antibody concentration in the tumor does not increase linearly with antibody dose; (e) raising the rate of antibody metabolism results in low concentration and poor percolation; (f) perhaps most interesting, there is predicted to be a range of antibody dose and affinity within which the specificity ratio and average concentration could be kept high while limiting the heterogeneity of distribution. PERC, the computer program package developed for these analyses, provides a convenient and flexible way to assess the impact of global and microscopic parameters on the distribution of immunoglobulin in tumors. For calculations presented here, the input data were obtained from experimental sources, and qualitative features of the output proved consistent with the few interpretable observations available. However, detailed validation would require much more data than are currently at hand. The mathematical findings should therefore be considered as aids to concept development and as a set of null hypotheses with which to guide experimentation. Experiments and simulations will continue in tandem. It should be noted that the PERC package (and also the general principles delineated here) can be applied as well to biological ligands other than antibodies.