S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution-glutamic acid to alanine at position 35-in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence ofthe smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.The generation ofantibody diversity has long been and remains one of the intriguing questions in immunology. Protein sequence analyses (1, 2) and nucleic acid studies (3-7) have similarly suggested that the number oflight (L) and heavy (H) chain genes in the germ line is large (>200 each). If random combinations of L and H chains were to occur, > 10,000 different antibodies could be generated solely from the germ-line repertoire. Furthermore, immunoglobulin chains are encoded in multiple genetic elements. The variable domains oflight chains are encoded by two gene segments designated variable (V) and joining (J) (8, 9). Heavy chain variable domains, in addition to V and J segments, have a third element, D (diversity), that encodes a portion of the third hypervariable region (10-12). The combination of a given L chain V gene with any of four functional J genes can thus produce additional structural diversity as can V, D, and J recombination in the H chain. The potential sequence diversity is further increased by variations in the sites at which these elements combine (8,9,(13)(14)(15)(16)(17). At present, it is not clear how much the sequence diversity generated by these events contributes to functional changes that affect the specificity and affinity of antigen binding.In view of the large amount of structural diversity that can be generated from the germ-line repertoire and the recombination events occurring during the formation of active immunoglobulin genes, the question of the occurrence and role of somatic mutation in the generation of antibody diversity remains to be determined. The initial studies ofmouse A L chains by Weigert and co-workers (18, 19) identified 12 invariant sequences, 5 with single amino acid substitutions, 1 with two substitutions, and 1 with three substitutions. All interchanges were located in hypervariable regions and it was concluded that the variants arose by a so...

SummaryMice with transgenes containing an antibody H chain V region (V.DJ.) gene were used in an analysis of the c/s-acting elements required for hypermutation of immunoglobulin (Ig) V genes. These transgenes can somatically recombine with endogenous IgH DNA, leading to the formation of functional heavy (H) chains partially encoded by the transgenic VsDJ.. The transgenomes in the five different lines of mice analyzed contain as little as 150 bp, and as much as 2.8 kb of natural DNA flanking the 5' side of the V. and either 1.5 or 2.3 kb (including the intronic enhancer and 5' matrix attachment region [MAR]) flanking the 3' side of Vs. Hybridomas were constructed from immunized transgenic mice, and transgenes present in these hybridomas that had or had not recombined to form functional H chain loci were sequenced. The data obtained show that: (a) the recombined transgenes contain hypermutated V. genes; and (b) among such transgenes, even those containing only 150 bp of natural V. 5' flanking sequence and several kilobases of 5' plasmid vector sequence display a frequency, distribution, and type of mutation characteristic of conventional IgH loci. The data also indicate that transgenic VsDJ. genes that have not recombined with endogenous IgH DNA are not substrates for hypermutation, even if they are flanked by 2.8 kb of natural 5' DNA, and 2.3 kb of natural 3' DNA, including the J.2-JH4 region, a MAR, and the intronic enhancer. Collectively, the data suggest that sequences 5' of the V. promoter are dispensable, a V. promoter and the intronic IgH enhancer region are not sufficient, and a region(s) within or 3' of the IgH constant region locus is requisite, for hypermutafion of Ig V. transgenes. ntibody V region diversity in mice and humans is generated by the combinatorial joining of germline V gene segments that are members of heterogeneous multigene families, the deletion and de novo addition of nucleotides at the junctions of these V segments during joining, and hypermutation of the resulting V genes (1). While characterization of the cis-and trans-acting factors involved in the rearrangement of V gene segments and the generation of junctional sequences has proceeded rapidly in recent years (2, 3), the mechanism of hypermutation continues to remain enigmatic.Current evidence suggests that hypermutation is induced during an immune response (4-6), introduces mainly singlenucleotide replacements at a rate estimated to be 10 .3 per base pair per cell division (7), acts efficiently only in and immediately around fully rearranged V genes (8-14), and is not causally linked to isotype switching (15, 16). Since V gene hypermutation plays a central role in the affinity maturation of antibodies (6,17,18), and is intimately associated with the development of B cell memory (17,(19)(20)(21)(22)(23), the complete elucidation of its mechanism, as well as how this mechanism is regulated, is of central importance to an understanding of the development of antibody specificity and humoral immunity.While direct approaches to the cha...

Background: Maternity care practices such as skin-to-skin care, rooming-in, and direct breastfeeding are recommended, but it is unclear if these practices increase the risk of clinically significant COVID-19 in newborns, and if disruption of these practices adversely affects breastfeeding. Methods: We performed a retrospective cohort study of 357 mothers and their infants <12 months who had confirmed or suspected COVID-19. Subjects came from an anonymous worldwide online survey between May 4 and September 30, 2020, who were recruited through social media, support groups, and health care providers. Using multivariable logistic regression, Fisher's exact test, and summary statistics, we assessed the association of skin-to-skin care, feeding, and rooming-in with SARS-CoV-2 outcomes, breastfeeding outcomes, and maternal distress. Results: Responses came from 31 countries. Among SARS-CoV-2+ mothers whose infection was £3 days of birth, 7.4% of their infants tested positive. We found a nonsignificant decrease in risk of hospitalization among neonates who roomed-in, directly breastfed, or experienced uninterrupted skin-to-skin care (p > 0.2 for each). Infants who did not directly breastfeed, experience skin-to-skin care, or who did not room-in within arms' reach, were significantly less likely to be exclusively breastfed in the first 3 months, adjusting for maternal symptoms (p £ 0.02 for each). Nearly 60% of mothers who experienced separation reported feeling ''very distressed,'' and 29% who tried to breastfeed were unable. Presence of maternal symptoms predicted infant transmission or symptoms (adjusted odds ratio = 4.50, 95% confidence interval = 1.52-13.26, p = 0.006). Conclusion: Disruption of evidence-based quality standards of maternity care is associated with harm and may be unnecessary.

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Transgenic lines of mice were derived by using plasmid constructs containing DNA encoding an antibody heavy chain variable-diversity-joining region (VH-D-JH) and various amounts of 5' and 3' flanking DNA but lacking any repetitive isotype switch (S) or constant (C) region DNA.Unexpectedly, many of the antibody VH regions expressed by B-ceil hybridomas generated from immunized transgenic mice were found to be of transgenic origin. Further analyses showed that somatic events had generated hybrid genomic loci in the mice containing the transgenic VH-D-JH gene and plasmid sequences 5' of endogenous heavy chain C region genes. Thus, VH-D-JH transgenes lacking S and C region DNA can recombine with endogenous Igh DNA, leading to the expression of transgene-encoded antibody.Antibody variable (V) region diversity in mice is generated by the combinatorial joining of germ-line V gene segments that are members of heterogeneous multigene families; the deletion and de novo addition of nucleotides at the junctions of these V gene segments during joining; and hypermutation of the resulting V genes (1). During the immune response of strain A/J mice to the hapten p-azophenylarsonate (Ars), antibody V regions encoded by a single combination of V gene segments (termed "canonical") become predominant and hypermutated (2). As part of an investigation of the cis-acting elements that direct the hypermutation process to antibody V genes, we generated transgenic mice using plasmid vectors containing a canonical heavy chain V gene (VH). MATERIALS AND METHODSTransgenic Mice. Transgenic mice were produced by using published protocols (3). Fertilized eggs were from matings of C57BL/6 x A/J F1 female mice and A/J male mice.Immunization and Generation of Hybridomas. Mice were injected i.p. with 100 ,ug of Ars-conjugated keyhole limpet hemocyanin (Ars-KLH) in complete Freund's adjuvant. One week later three i.p. injections of 100 pug of Ars-KLH in phosphate-buffered saline were given at 3-day intervals. Three days after the final injection, spleen cells were used in fusions to Sp2/0 cells as described (4). Idiotype and isotype assays on supernatants were done as described (4). DNA Isolation and Southern Blot Analysis. DNA was isolated from hybridomas, purified A phage, tails, or spleens as described (5). Sequential Southern blotting analyses were done (4) with the following probes: pBluescript KS(-) (Stratagene); VhUp [an Xba I-Pst I fragment specific for sequences upstream of the A/J anti-Ars hybridoma 36-65 (6) VH coding region ending within the leader exon]; Vh133 [an Ava II-Rsa I fragment encompassing codons 15-59 in the hybridoma 36-65 VH gene (7)]; an Xba I-EcoRI fragment containing the Igh enhancer; J14B, a HindIII fragment 5' of the enhancer and 3' ofthe A heavy chain switch (S) region (8); pyl/EH10.0 specific for yl heavy chain S-region DNA (Sl) (9); pyl/A5, specific for the yl chain constant (C) coding region (Cr1) (10); and py2b/E6.6, specific for Sy2b DNA (9).Serology. Normal A/J and transgenic mice were immunized i.p. with ...

The SAP rate observed in procedures with SAP indication and the appropriateness of drug choice, timing, and duration are reasons of concern. Quality improvement interventions for implementing SAP recommendations in children are strongly needed, and their impact should be evaluated at hospital level.

The S107 mouse myeloma cell line synthesizes an IgA antibody that binds the hapten phosphocholine and is similar ifnot identical in its heavy and light chain variable region sequence to the predominant antibody produced by BALB/c mice in response to immunization with phosphocholine. This cell line frequently and spontaneously generates somatic variants producing immunoglobulins with decreased ability to bind antigen. One such variant, S107.U1, is described here. This variant has a decreased ability to bind phosphocholine when it is attached to a carrier, although its affinity for free hapten is the same as that of the parent. This decrease in antigen binding is associated with a single amino acid substitution at the fifth residue in the JH segment.The many recent studies on the structure of the genes coding for immunoglobulin heavy (H) and light (L) chains have provided new insights into some ofthe mechanisms responsible for the generation of antibody diversity. The enormous sequence diversity of mouse K L chains is to a large degree explained by the random recombination between a few hundred germ line variable (V) region genes and one of the four joining (J) minigenes that code for the COOH-terminal 13 amino acids of the V region (1-3). Further diversification is possible and has been found to occur through variation in the V-J recombination site (4-6). Even greater sequence diversity can be achieved in H chains through random recombination between any one of the many germ line V region genes, a minigene called D (diversity) that is located between V and J and codes for a portion of the third hypervariable region, and the H chain J segments (7,8). As with K L chains, variation in the recombination sites between germ line V, D, andJ will result in additional sequencediversity (5,6). However, these somatic recombinational events account for only some of the sequence diversity in the third hypervariable regions of the H and L chains. More importantly, they do not provide an explanation for all of the diversity observed in the first and second hypervariable regions as well as the first three framework regions of the H and L chains, which are encoded in a continuous linear sequence in the germ line genes (9, 10).One approach to understanding the sequence diversity in the first and second hypervariable regions has been to determine or deduce the germ line sequence of a particular H or L chain V region gene and then to analyze-the progeny of this gene as they are expressed as antibody molecules in the form of myeloma or hybridoma monoclonal antibodies. This form of pedigree analysis (11) with the mouse A L chains led Weigert and Cohn and their colleagues to suggest that a somatic mechanism resembling mutation was responsible for the sequence diversity in the first and second hypervariable regions (12,13 (17) provide strong support for the importance of a somatic mutational mechanism in the generation of the structural diversity of antibodies.

Somatic hypermutation and selection of immunoglobulin (Ig) variable (V)-region genes, working in concert, appear to be essential for memory B-cell development in mammals. There has been substantial progress on the nature of the cis-acting DNA elements that regulate hypermutation. The data obtained suggest that the mechanisms of Ig gene hypermutation and transcription are intimately intertwined. While it has long been appreciated that stringent phenotypic selection forces are imposed on the somatically mutated Ig V regions generated during a T-cell dependent B-cell response, the mechanisms involved in this selection have remained enigmatic. Our studies have questioned the role of foreign antigen deposited on follicular dendritic cells in affinity-based positive selection of V regions, and have shown that this selection takes place in a "clone-autonomous" fashion. In addition, our data strongly suggest that affinity for antigen alone is not the driving force for selection of B-cell clones into the memory compartment. In contrast, we suggest that a combination of positive selection for increased foreign antigen binding, and negative selection of antibody V regions that are autoreactive at the onset of the response, or have acquired autoreactivity via hypermutation, results in the "specificity maturation" of the memory B-cell response.