Internal medicine fellowship directors' perspectives on the quality and utility of letters conforming to residency program director letter of recommendation guidelines.

Background: In May 2017, the Alliance for Academic Internal Medicine (AAIM) published guidelines intending to standardize and improve internal medicine residency program director (PD) letters of recommendation (LORs) for fellowship applicants. Objectives: This study aimed to examine fellowship PDs impressions of the new guidelines, letter writers' adherence to the guidelines, and the impact of LORs that conformed to guidelines compared to non-standardized letters. Methods: The authors anonymously surveyed fellowship PDs from January to March 2018 to gather input about LORs submitted to their programs during the 2017 fellowship application cycle. Results: A total of 78% of survey respondents were satisfied with letters that followed the AAIM guidelines, whereas 48% of respondents were satisfied with letters that did not. Fellowship PDs felt that letters that followed the AAIM guidelines were more helpful than letters that did not, especially for differentiating between applicants from the same institution and for understanding residents' performance across the six core competency domains. Fellowship PDs provided several suggestions for residency PDs to make the LORs even more helpful. Conclusion: Fellowship PD respondents indicated that LORs that followed the new AAIM guidelines were more helpful than letters that did not.

Chimpanzee models for human disease and immunobiology.

Chimpanzees have greater than 98% genomic sequence homology with humans but have significantly more favorable responses to human immunodeficiency virus (HIV)-1 and hepatitis B virus (HBV) and an apparently low incidence of epithelial malignancy. Although there are few shared major histocompatibility complex (MHC) alleles between human and chimp, there is considerable overlap in binding repertoires for epitopes of HIV-1 and HBV. This indicates that differences in viral handling may be due to involvement of cells other than T lymphocytes. Similar mechanisms may be involved in host response to dysplastic or malignant cells. In seeking to understand these differences, most attention has been focused on comparing and contrasting well-characterized steps in immune response. As an additional possibility, alterations in cell-cell interactions dependent upon sialic acid binding proteins known to be involved in immune responses should also be considered. The lack of a particular sialic acid structure (N-glycolyl neuraminic acid, or Neu5Gc) in humans, due to a gene mutation in an essential synthetic enzyme, has potentially altered the kinetics of cellular responses dependent upon these lectins. The absence of Neu6Gc represents the only known major biochemical difference between humans and chimpanzees.

A structural difference between the cell surfaces of humans and the great apes.

The sialic acids are major components of the cell surfaces of animals of the deuterostome lineage. Earlier studies suggested that humans may not express N-glycolyl-neuraminic acid (Neu5Gc), a hydroxylated form of the common sialic acid N-acetyl-neuraminic acid (Neu5Ac). We find that while Neu5Gc is essentially undetectable on human plasma proteins and erythrocytes, it is a major component in all the four extant great apes (chimpanzee, bonobo, gorilla and orangutan) as well as in many other mammals. This marked difference is also seen amongst cultured lymphoblastoid cells from humans and great apes, as well as in a variety of other tissues compared between humans and chimpanzees, including the cerebral cortex and the cerebrospinal fluid. Biosynthetically, Neu5Gc arises from the action of a hydroxylase that converts the nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc. This enzymatic activity is present in chimpanzee cells, but not in human cells. However, traces of Neu5Gc occur in some human tissues, and others have reported expression of Neu5Gc in human cancers and fetal tissues. Thus, the enzymatic capacity to express Neu5Gc appears to have been suppressed sometime after the great ape-hominid divergence. As terminal structures on cell surfaces, sialic acids are involved in intercellular cross-talk involving specific vertebrate lectins, as well as in microbe-host recognition involving a wide variety of pathogens. The level of sialic acid hydroxylation (level of Neu5Ac versus Neu5Gc) is known to positively or negatively affect several of these endogenous and exogenous interactions. Thus, there are potential functional consequences of this widespread structural change in humans affecting the surfaces of cells throughout the body.

A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence.

Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts of N-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acid N-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase cDNAs. Although this chimpanzee cDNA is similar to the murine homologue, the human cDNA contains a 92-bp deletion resulting in a frameshift mutation. The isolated human gene also shows evidence for this deletion. Genomic PCR analysis indicates that this deletion does not occur in any of the African great apes. The gene is localized to 6p22-p23 in both humans and great apes, which does not correspond to known chromosomal rearrangements that occurred during hominoid evolution. Thus, the lineage leading to modern humans suffered a mutation sometime after the common ancestor with the chimpanzee and bonobo, potentially affecting recognition by a variety of endogenous and exogenous sialic acid-binding lectins. Also, the expression of Neu5Gc previously reported in human fetuses and tumors as well as the traces detected in some normal adult humans must be mediated by an alternate pathway.

Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.

Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.

Developmental sialic acid modifications in rat organs.

The changes in expression of sialic acids in Sprague-Dawley rats in the prenatal and early postnatal time period have been examined in multiple organs, both visceral and non-visceral. In all organs examined, there is a dramatic increase in both N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) shortly after birth. The bulk of the sialic acid is present in the ganglioside fraction in all tissues examined. As total amounts of sialic acid present in gangliosides decrease, the proportion present in the low molecular weight cytosolic fraction increases. A curious observation is that Neu5Ac hydroxylase activity is present at the time of the increase in sialic acid, but its activity does not correlate with Neu5Gc expression after the early postnatal period. This implies that Neu5Gc expression has another level of regulation besides CMP-Neu5Ac hydroxylase activity.

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool.

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.

Developmental regulation of sialic acid modifications in rat and human colon.

Using high-pressure liquid chromatography (HPLC) and gas-liquid chromatography/mass spectrometry (GLC/MS), we have confirmed the existence of several sialic acid modifications in the adult rat and human colon. The major O-acetylated sialic acid in both species is 9-O-acetyl-N-acetylneuraminic acid; N-glycolylneuraminic acid is a major component of the adult rat colon. Both of these major modifications were found to be developmentally regulated during the perinatal period in the rat. The N-glycolyl modification is present prenatally and disappears rapidly in the postnatal period. It reappears in the preweanling period, reaching levels at weaning comparable to those found prenatally. In contrast, the 9-O-acetyl modification is very low prenatally, and undergoes a marked increase shortly after birth in both the rat and human colon. The difference in the kinetics of appearance of the two modifications suggests that they are independently regulated. Regulation of these modifications seems to be influenced by exposure to bacterial by-products or environmental stimuli. The N-glycolyl modification in the rat colon reappeared at weaning, a time of major change in enteral colonic substances. Spontaneously aborted human fetuses, including three with intrauterine infection at 27, 33, and 35 wk of gestation, showed adult levels of O-acetylation in colonic tissue. Also, although O-acetylation in freshly isolated colon tumor specimens was only somewhat lower than that in the adult normal colon, all established colon cancer cell lines studied showed minimal O-acetylation.

Selective inactivation of influenza C esterase: a probe for detecting 9-O-acetylated sialic acids.

The influenza C virus (INF-C) hemagglutinin recognizes 9-O-acetyl-N-acetylneuraminic acid. The same protein contains the receptor-destroying enzyme (RDE), which is a 9-O-acetyl-esterase. The RDE was inactivated by the serine esterase inhibitor di-isopropyl fluorophosphate (DFP). [3H]DFP-labeling localized the active site to the heavy chain of the glycoprotein. DFP did not alter the hemagglutination or fusion properties of the protein, but markedly decreased infectivity of the virus, demonstrating that the RDE is important for primary infection. Finally, DFP-treated INF-C bound specifically and irreversibly to cells expressing 9-O-acetylated sialic acids. This provides a probe for a molecule that was hitherto very difficult to study.

The role of antigen mobility in anti-Rh0(D)-induced agglutination.

Intact human erythrocytes were cross-linked with glutaraldehyde (GA) or dimethyladipimidate (DMA) and tested for their ability to bind [125I]-IgG anti-Rh0(D) and to undergo antibody-mediated hemagglutination. There was no decrease in antibody binding after treatment with GA concentrations up to 1.25% and DMA concentrations up to 1%. Red cells treated with these concentrations of GA and DMA did not agglutinate. The techniques employed to induce agglutination of the cross-linked red cells involved "incomplete" IgG anti-Rho (D) in albumin, "complete" IgM anti-D Rho (D) in saline, and the antiglobulin (Coombs) reaction. The agglutinability of the chemically modified red cells was inversely correlated with the extent of fixation. The dissociation of antibody binding from agglutinability in cross-linked erythrocytes suggests that Rho (D) antigen mobility is required for red cell agglutination. Antigen mobility was manifested by the transition from a relatively monodisperse distribution pattern of Rho (D) antigen sites to one of large aggregates or clusters when agglutination was induced by IgM anti-Rho (D), IgG anti-Rho (D) agglutination of protease modified red cells, and by anti-IgG agglutination of IgG anti-Rho (D)-coated red cells. Antigen clustering was not as prominent in red cells agglutinated by IgG anti-Rho (D) in the presence of albumin. Even though antigen mobility is a prerequisite for antibody-mediated hemagglutination, clustering does not appear to be an absolute requirement. The degree of antigen clustering differs with varying types of agglutination.