A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.     

In vitro test systems supporting the development of improved pest control methods: a case study with chemical mixtures and bivalve biofoulers

Using the bivalve macrofouler Corbicula fluminea, the suitability of in vitro testing as a stepping stone towards the improvement of control methods based on chemical mixtures was addressed in this study. In vitro cholinesterase (ChE) activity inhibition following single exposure of C. fluminea tissue to four model chemicals (the organophosphates dimethoate and dichlorvos, copper and sodium dodecyl phosphate [SDS]) was first assessed. Consequently, mixtures of dimethoate with copper and dichlorvos with SDS were tested and modelled; mixtures with ChE revealed synergistic interactions for both chemical pairs. These synergic combinations were subsequently validated in vivo and the increased control potential of these selected combinations was verified, with gains of up to 50% in C. fluminea mortality relative to corresponding single chemical treatments. Such consistency supports the suitability of using time- and cost-effective surrogate testing platforms to assist the development of biofouling control strategies incorporating mixtures.     

Formation and reversible dissociation of coiled coil of peptide to the C-terminus of the HSV B5 protein: a time-resolved spectroscopic analysis

An understanding of the molecular mechanisms of the newly characterized herpes simplex virus (HSV) B5 protein is important to further elucidate the HSV cell entry and infection. The synthetic peptide of B5 (wtB5) was functionalized with the nonlinear optical chromophore cascade yellow and its molecular dynamics was probed at physiological and endosomal pH (pH 7.4 and 5.5, respectively). Steady-state CD spectroscopy was utilized to characterize the peptides at different pH. These spectra showed structural changes in the peptide with time measured over several days. Nonlinear optical measurements were carried out to probe the interactions and local environment of the labeled peptide, and the increase in the two-photon cross section of this system suggests an increase in chromophore-peptide interactions. Time-resolved fluorescence upconversion measurements reflected changes in the hydrophilic and hydrophobic local environments of the labeled peptide-chromophore system. Ultrafast depolarization measurements gave rotational correlation times indicative of a reversible change in the size of the peptide. The time-resolved results provide compelling evidence of a reversible dissociation of the coiled coils of the wtB5 peptide. This process was found to be pH-insensitive. The data from this unique combination of techniques provide an initial step to understanding the molecular dynamics of B5 and a framework for the development of novel imaging methods based on two-photon emission, as well as new therapeutics for HSV.     

Continuous Metabolic Syndrome Scores for Children Using Salivary Biomarkers

Background

Binary definitions of the metabolic syndrome based on the presence of a particular number of individual risk factors are limited, particularly in the pediatric population. To address this limitation, we aimed at constructing composite and continuous metabolic syndrome scores (cmetS) to represent an overall measure of metabolic syndrome (MetS) in a large cohort of metabolically at-risk children, focusing on the use of the usual clinical parameters (waist circumference (WC) and systolic blood pressure (SBP), supplemented with two salivary surrogate variables (glucose and high density lipoprotein cholesterol (HDLC). Two different approaches used to create the scores were evaluated in comparison.

Methods

Data from 8,112 Kuwaiti children (10.00 ± 0.67 years) were used to construct two cmetS for each subject. The first cmetS (cmetS-Z) was created by summing standardized residuals of each variable regressed on age and gender; and the second cmetS (cmetS-PCA) was defined as the first principal component from gender-specific principal component analysis based on the four variables.

Results

There was a graded relationship between both scores and the number of adverse risk factors. The areas under the curve using cmetS-Z and cmetS-PCA as predictors for severe metabolic syndrome (defined as the presence of ≥3 metabolic risk factors) were 0.935 and 0.912, respectively. cmetS-Z was positively associated with WC, SBP, and glucose, but inversely associated with HDLC. Except for the lack of association with glucose, cmetS-PCA was similar to cmetS-Z in boys, but had minimum loading on HDLC in girls. Analysis using quantile regression showed an inverse association of fitness level with cmetS-PCA (p = 0.001 for boys; p = 0.002 for girls), and comparison of cmetS-Z and cmetS-PCA suggested that WC and SBP were main contributory components. Significant alterations in the relationship between cmetS and salivary adipocytokines were demonstrated in overweight and obese children as compared to underweight and normal-weight children.

Conclusion

We have derived continuous summary scores for MetS from a large-scale pediatric study using two different approaches, incorporating salivary measures as surrogate for plasma measures. The derived scores were viable expressions of metabolic risk, and can be utilized to study the relationships of MetS with various aspects of the metabolic disease process.     

Effects of an abrupt diet change from hay to concentrate on microbial numbers and physical environment in the cecum of the pony.

Microbial numbers, pH, fluid volume, and turnover rate in the pony cecum were measured during an abrupt change from an all-forage to an all-concentrate diet, both fed at maintenance energy levels. Concentrate feeding resulted in increased (P less than 0.01) numbers of total viable anaerobic bacteria. The numbers of organisms growing on selective starch medium increased (P less than 0.01) when concentrate was fed, while numbers on xylan and pectin media decreased (P less than 0.025). Seven days after the diet change to concentrate, the number of bacteria growing on lactate medium increased (P less than 0.01), followed by a gradual decline. Cellulolytic bacteria occurred in low numbers, ranging from 1.1 x 10(4) to 4.4 x 10(4) per g of cecal contents. Feeding all concentrate decreased both the number of genera (P less than 0.01) and total protozoan numbers (P less than 0.01) in the cecum. Minimum cecal pH values of 6.4 and 5.8 were obtained when forage and concentrate, respectively, were fed, with the minimum pH occurring 6 h postfeeding. Dry-matter percentage of cecal contents followed a diurnal pattern which was the inverse of the pH curve. During forage feeding, the cecum contained an average of 2.2 liters (1.6 to 3.4 liters), which turned over 3.9 times per day. When concentrate was fed, cecal volume averaged 3.9 liters (0.6 to 8.6 liters), with a mean liquid turnover of 4.2 times per day. Microbial numbers and pH changes in the pony cecum associated with an abrupt change in diet from hay to concentrate resembled those which occur in the rumen under similar feeding conditions.     

The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli

R. J. ROWBURY, M. GOODSON AND A.D. WALLACE. 1992. Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0. Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH. The habituation sensor does not detect increased ΔpH (or decreased Δψ) nor an increased difference between pH_o and periplasmic pH but probably detects a fall in either external or periplasmic pH. Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions. Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant. Strains derepressed for phoE , in contrast, showed increased acid sensitivity. These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions. Stimulation by phosphate of the poor growth of E. coli at pH 5.0 is in accord with the above. Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pH_i near to neutrality at an acidic pH_o level.     

Alcoholism and substance abuse among selected southern cheyenne Indians

This family and small community-based study reports the occurrence of alcoholism and co-occurring substance abuse in Southern Cheyenne Indians living in western Oklahoma. Sociocultural factors complicate operationalization of clinical data into standard (DSM-III-R) psychiatric disorder terminology; understanding sociocultural factors is essential for assessing the high rate of addictive disorders in this group. To obtain reliable and valid clinical diagnoses, data from several sources were utilized within a blind rating system: 1) SADS-L, a clinician-administered research diagnostic instrument; 2) MAST; 3) relatives; 4) medical records; 5) other official documents. The sample consisted of 69 males (45 alcoholics) and 97 females (36 alcoholics). Among clinically significant substance abusers (moderate impairment of function), 22 of 24 were alcoholics. In non-alcoholics, mean MAST scores were 8.8 (males) and 5.1 (females); in alcoholics, 32.0 (males) and 38.7 (females). Mean age of onset on heavy use of alcohol was 20.1 yrs. (males) and 22.8 (females) (p = 0.047); among all alcoholics, 86% (males) and 64% (females) had early onset (< 25 yrs. old). When data from 98 unrelated subjects were analyzed separately, similar findings were observed except that mean age of onset of heavy use of alcohol was more discrepant between males and females, viz. 20.1 versus 22.8 yrs. (p = 0.02). Among those with substance abuse disorders, early age of onset was present in all but one female. In these Cheyenne, alcoholism is usually clinically severe and early in onset; it often co-occurs with substance abuse, also early in onset.     

The PhoE porin and transmission of the chemical stimulus for induction of acid resistance (acid habituation) in Escherichia coli.

Escherichia coli K12 becomes resistant to killing by acid (habituates to acid) in a few minutes at pH 5.0. Habituation involves protein synthesis-dependent and -independent stages; both must occur at an habituating pH. The habituation sensor does not detect increased delta pH (or decreased delta psi) nor an increased difference between pHo and periplasmic pH but probably detects a fall in either external or periplasmic pH. Phosphate ions inhibit habituation, at any stage, probably by interfering with outer membrane passage of hydrogen ions. Most outer membrane components tested are not required for habituation but phoE deletion mutants habituated poorly and are acid-resistant. Strains derepressed for phoE, in contrast, showed increased acid sensitivity. These and other results suggest that habituation involves hydrogen ions or protonated carriers crossing the outer membrane preferentially via the PhoE pore, a process inhibited by phosphate and other anions. Stimulation by phosphate of the poor growth of E. coli at pH 5.0 is in accord with the above. Acetate did not enhance acid killing of pH 5.0 cells, suggesting that their resistance does not depend on maintaining pHi near to neutrality at an acidic pHo level.