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SummaryLate-onset Alzheimer’s Disease (LOAD) pathology is rare in our closest living evolutionary relatives (chimpanzees), which also express much lower microglial levels of CD33(Siglec-3)–a myelomonocytic receptor inhibiting innate immune reactivity by extracellular V-set domain recognition of sialic acid(Sia)-containing “self-associated molecular patterns” (SAMPs). We earlier showed that V-set domain-deficient CD33-variant allele, protective against LOAD, is derived and specific to hominin-lineage. We now report that CD33 also harbors multiple hominin-specific V-set domain mutations and explore selection forces that may have favored such genomic changes. N-glycolylneuraminic acid (Neu5Gc), the preferred Sia-ligand of ancestral CD33 is absent in humans, due to hominin-specific, fixed loss-of-function mutation in CMAH, which generates CMP-Neu5Gc from its precursor, CMP-N-acetylneuraminic acid (Neu5Ac). Extensive mutational analysis and MD-simulations indicate that fixed change in amino acid 21 of hominin V-set domain and conformational changes related to His45 corrected for Neu5Gc-loss by switching to Neu5Ac-recognition. Considering immune-evasive “molecular mimicry” of SAMPs by pathogens, we found that human-specific pathogens Neisseria gonorrhoeae and Group B Streptococcus (affecting fertility and fetuses/neonates respectively) selectively bind huCD33 and this binding is significantly impacted by amino acid 21 modification. Alongside LOAD-protective CD33 alleles, humans harbor additional, derived, population-universal, cognition-protective variants absent in “great ape” genomes. Interestingly, 11 of 13 SNPs in these human genes (including CD33), that protect the cognitive health of elderly populations, are not shared by genomes of archaic hominins: Neanderthals and Denisovans. Finally, we present a plausible evolutionary scenario to compile, correlate and comprehend existing knowledge about huCD33 evolution and suggest that grandmothering emerged in humans.

Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as nonproductive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined because of a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBCs) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with the current view of mucins as providing a protective shield against pathogens. Counterintuitively, increasing the density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.

SUMMARYFor a sperm to successfully fertilize an egg, it must first undergo capacitation in the female reproductive tract, and later undergo acrosomal reaction (AR) upon encountering an egg surrounded by its vestment. How premature AR is avoided despite rapid surges in signaling cascades during capacitation remains unknown. Using a combination of KO mice and cell-penetrating peptides, we show that GIV (CCDC88A), a guanine nucleotide-exchange modulator (GEM) for trimeric GTPases, is highly expressed in spermatocytes and is required for male fertility. GIV is rapidly phosphoregulated on key tyrosine and serine residues in human and murine spermatozoa. These phosphomodifications enable GIV-GEM to orchestrate two distinct compartmentalized signaling programs in the sperm tail and head; in the tail, GIV enhances PI3K→ Akt signals, sperm motility and survival, whereas in the head it inhibits cAMP surge and premature AR. Furthermore, GIV transcripts are downregulated in the testis and semen of infertile men. These findings exemplify the spatiotemporally segregated signaling programs that support sperm capacitation and shed light on a hitherto unforeseen cause of infertility in men.GRAPHIC ABSTRACTHIGHLIGHTSGIV is highly expressed in spermatozoa, and is required for male fertilityGIV is rapidly phosphoregulated during sperm capacitationIt enhances tyrosine-based signals in sperm tail, enhances motilityIt suppresses cAMP in the sperm head, inhibits premature acrosome exocytosis

Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as non-productive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined due to a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBC) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with current view of the mucins as providing a protective shield against pathogens. Counterintuitively, increasing density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell surface. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.SignificanceLike other animal hosts, humans are constantly challenged by pathogens. This has led to an evolution of physical barriers coating all mucosal tissues, which are most vulnerable to infection. An important part of this defense is a dense brush of large proteins, called mucins, which are heavily decorated with sugars and keep pathogens at bay. Deciphering how pathogens overcome the mucin barrier is necessary to understand early stages of infection and to develop more effective treatments. By artificially installing the mucin-like shield on the surfaces of cells using synthetic sugar-bearing polymers, we have discovered a new physical mechanism by which proteins and viruses can exploit this barrier to more strongly adhere to their targets.

Author(s): Smith, Meghan B; Ho, Jacqueline; Ma, Lihong; Lee, Miryoung; Czerwinski, Stefan A; Glenn, Tanya L; Cool, David R; Gagneux, Pascal; Stanczyk, Frank Z; McGinnis, Lynda K; Lindheim, Steven R | Abstract: ObjectiveTo examine the changes in AMH levels longitudinally over time and their relationship with both body composition, particularly abdominal adiposity, and milestones of pubertal development in female children.DesignSecondary analysis of a prospective, longitudinal study.SettingUniversity affiliated research center and laboratories.PatientsEighty-nine females were examined between 1990 and 2015 to study child growth and development.InterventionsDemographic, anthropometric, growth, and pubertal milestone data with serum samples stored and subsequently analyzed for AMH.Main outcome measuresLongitudinal change in AMH and predicted AMH levels based on body composition, age, and pubertal milestones including, pubarche, thelarche, and menarche.ResultsNatural log-transformed AMH (AMHlog) levels appeared to have a nonlinear relationship with age, decreasing between 10 and 14 years of age, increasing until 16 years. A mixed effect linear model demonstrated that increased abdominal adiposity (waist/height ratio, WHtR) was significantly associated with the predicted increased AMHlog levels (β=1.37). As females progressed through the Tanner stages, the model predicted decreasing AMHlog values when adjusting for age and WHtR.ConclusionsDeclining AMH levels during puberty may not be reflective of diminished ovarian reserve as observed in adults, but may suggest a permissive role of AMH in the activation of the hypothalamic-pituitary-ovarian axis.

Humans and orcas are among the very rare species that have a prolonged post-reproductive lifespan (PRLS), during which the aging process continues. Reactive oxygen species (ROS) derived from mitochondria and from the NADPH oxidase (NOX) enzymes of innate immune cells are known to contribute to aging, with the former thought to be dominant. CD33-related-Siglecs are immune receptors that recognize self-associated-molecular-patterns and modulate NOX-derived-ROS. We herewith demonstrate a strong correlation of lifespan with CD33rSIGLEC gene number in 26 species, independent of body weight or phylogeny. The correlation is stronger when considering total CD33rSIGLEC gene number rather than those encoding inhibitory and activating subsets, suggesting that lifetime balancing of ROS is important. Combining independent lines of evidence including the short half-life and spontaneous activation of neutrophils, we calculate that even without inter-current inflammation, a major source of lifetime ROS exposure may actually be neutrophil NOX-derived. However, genomes of human supercentenarians (>110years) do not harbor a significantly higher number of functional CD33rSIGLEC genes. Instead, lifespan correlation with CD33rSIGLEC gene number was markedly strengthened by excluding the post-reproductive lifespan of humans and orcas (R2 =0.83; P&nbsp

Cesarean scar ectopic pregnancy (CSEP) has a high rate of morbidity with nonspecific signs and symptoms making identification difficult. The criterion-standard treatment of CSEP has been subject to debate.This review defines CSEP, discusses pathogenesis and diagnosis, and compares treatment options and outcomes.A literature review was performed utilizing the termFive basic pathways have been identified in treatment of CSEP: expectant management, medical therapy, surgical intervention, uterine artery embolization, or a combination approach. Expectant management has the highest probability of morbid outcomes, including hemorrhage, uterine rupture, and preterm delivery. Medical management often requires further treatment with additional medication or surgery. Different surgical methods have been explored including uterine artery embolization; dilation and curettage; surgical removal via vaginal, laparoscopic, or laparotomic approach; and hysterectomy. Each method has various levels of success and depends on surgeon skill and patient presentation.Recent research supports any method that removes the pregnancy and scar to reduce morbidity and promote future fertility. Laparoscopic and transvaginal approaches are options for CSEP treatment, although continued research is required to identify the optimal approach.As cesarean delivery numbers rise, a subsequent increase in CSEPs can be anticipated. The ability to accurately diagnose and treat this morbid condition is vital to the practice of any specialist in general obstetrics and gynecology.

Humans can incorporate the xenoglycan N-glycolylneuraminic acid (Neu5Gc) from the diet into reproductive tissues and secretions. Most humans also have circulating antibodies specific for this dietary xenoglycan. The potential for inflammation induced by incorporated Neu5Gc and circulating anti-Neu5Gc antibodies, termed xenosialitis, has been discussed as a factor influencing several human diseases. Potential effects of xenosialitis on human fertility remain unknown. Here, we investigate possible adverse effects of the presence of Neu5Gc on sperm or endometrium combined with anti-Neu5Gc antibodies in semen or uterine secretions in a mouse model. We use Cmah(-/-) mice, humanized for Neu5Gc deficiency. We find that the viability, migration, and capacitation of sperm with incorporated Neu5Gc are negatively affected when these are exposed to anti-Neu5Gc antibodies. In addition, we find that after copulation, activated uterine neutrophils and macrophages show increased phagocytosis of sperm in the presence of anti-Neu5Gc antibodies via the complement receptor 3 (C3R) and Fcγ I/II/III (Fc receptor). Furthermore, Neu5Gc in endometrial cells combined with the presence of anti-Neu5Gc antibodies alters the receptivity and decidualization of endometrial explants. These studies provide mechanistic insights on how Neu5Gc on sperm and/or endometrium combined with anti-Neu5Gc antibodies in semen and uterine fluid might contribute to unexplained human infertility.

The practice of ovulation induction often falls to the reproductive endocrinology and infertility specialist. However, attitudes toward the evaluation and treatment of infertility has shifted among general obstetrician-gynecologists (OB-GYN). This review discusses the underlying scientific basis of anovulation and clinical guidelines regarding the use of different medications for the purpose of promoting follicular recruitment and ovulation for the general OB-GYN.

Deep infiltrating endometriosis (DIE) is a debilitating subset of endometriosis with an ill-defined set of symptoms and lacks specific diagnostic criteria to identify, leading to significant delays in treatment. Endometriosis itself is associated with chronic pain and infertility, leading to significant healthcare costs. A literature has shown that DIE is a non-progressive and curable disease. Currently, diagnosis is based purely on surgical diagnosis, which carriers a higher rate of morbidity, and fear of surgery or reluctance on behalf of provider, may delay diagnosis and thus treatment. Multiple attempts have been made to create an appropriate classification system mainly based on symptoms or imaging, however no one scheme has been successful to appropriately identify DIE. Research has now been geared towards the use of biomarkers, including blood, urine, peritoneal/follicular fluid, or endometrial tissue, to construct a diagnostic tool for DIE. This has the potential to create a less invasive mechanism for earlier identification and treatment of this curable disease.

The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer's dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer's disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin.

SUMMARY Mammalian spermatozoa are coated with a thick glycocalyx that is assembled during sperm development, maturation, and upon contact with seminal fluid. The sperm glycocalyx is critical for sperm survival in the female reproductive tract and is modified during capacitation. The complex interplay among the various glycoconjugates generates numerous signaling motifs that may regulate sperm function and, as a result, fertility. Nascent spermatozoa assemble their own glycans while the cells still possess a functional endoplasmic reticulum and Golgi in the seminiferous tubule, but once spermatogenesis is complete, they lose the capacity to produce glycoconjugates de novo. Sperm glycans continue to be modified, during epididymal transit by extracellular glycosidases and glycosyltransferases. Furthermore, epididymal cells secrete glycoconjugates (glycophosphatidylinositol‐anchored glycoproteins and glycolipids) and glycan‐rich microvesicles that can fuse with the maturing sperm membrane. The sperm glycocalyx mediates numerous functions in the female reproductive tract, including the following: inhibition of premature capacitation; passage through the cervical mucus; protection from innate and adaptive female immunity; formation of the sperm reservoir; and masking sperm proteins involved in fertilization. The immense diversity in sperm‐associated glycans within and between species forms a remarkable challenge to our understanding of essential sperm glycan functions. Mol. Reprod. Dev. 82: 635–650, 2015. © 2015 The Authors. Molecular Reproduction and Development published by Wiley Periodicals, Inc.

Objective: To assess the frequency of dietary xenoglycanNeu5Gc and antibodies in males and females and its impact on fertility. Design: Prospective study of semen, uterine lavage, and follicular fluid from subjects undergoing infertility evaluation or in vitro fertilization (IVF) and fertile controls. Setting: University based infertility program. Participants: Males (n=23) and females (n=27) undergoing semen analysis and saline infusion sonography as part of their diagnostic evaluation and 37 women undergoing IVF were compared to fertile male (n=15) and female (n=14) controls. Intervention: Neu5Gc was measured by affinity purified antibody staining on Western blots, flow cytometry, and by high performance liquid chromatography. Anti- Neu5Gc antibodies were determined by ELISA. Main parameters measured: Frequency and levels of Neu5Gc antigen within sperm and endometrial cells and antibodies in semen, uterine lavage, and follicular fluid. Semen quality and IVF outcomes were assessed between antigen and antibody positive and negative subjects. Results: In infertile subjects,Neu5Gc was detected in 26% of sperm and 54% of endometrial cells compared to 0% in male and 0% female controls. Anti-Neu5Gc antibodies were identified in 54% of seminal fluid, 41% in uterine lavage and 43% of follicular fluid samples. There were no differences in semen parameters, oocyte quality, and embryo development in the presence or absence of Neu5Gc antigen or antibody. However, clinical pregnancy rate was significantly lower in the presence of anti-Neu5Gc antibodies intrauterine lavage (0% vs. 54.5.0%, p

Author(s): Havemann, Logan M; Cool, David R; Gagneux, Pascal; Markey, Michael P; Yaklic, Jerome L; Maxwell, Rose A; Iyer, Ashvin; Lindheim, Steven R | Abstract: ObjectiveThe aim of the study was to review the current nomenclature and literature examining microbiome cytokine, genomic, proteomic, and glycomic molecular biomarkers in identifying markers related to the understanding of the pathophysiology and diagnosis of vulvodynia (VVD).Materials and methodsComputerized searches of MEDLINE and PubMed were conducted focused on terminology, classification, and "omics" variations of VVD. Specific MESH terms used were VVD, vestibulodynia, metagenomics, vaginal fungi, cytokines, gene, protein, inflammation, glycomic, proteomic, secretomic, and genomic from 2001 to 2016. Using combined VVD and vestibulodynia MESH terms, 7 references were identified related to vaginal fungi, 15 to cytokines, 18 to gene, 43 to protein, 38 to inflammation, and 2 to genomic. References from identified publications were manually searched and cross-referenced to identify additional relevant articles. A narrative synthesis of the articles was conducted; however, meta-analysis was not conducted because of substantial heterogeneity in the studies and limited numbers of control-matched studies.ResultsVarying definitions of VVD complicate a meta-analysis, and standard definitions will better allow for comparisons of studies and enhance the applicability of evidence to patient populations. Although data are still limited, genomic and molecular diagnostic testings continue to be investigated as potential tools for the diagnosis of VVD.ConclusionsStandardized nomenclature will allow for comparability of studies and progress in research related to the pathophysiology of VVD and to facilitate clinical decision making and treatment choices. Although the current understanding of the pathogenesis of VVD is limited, there are new opportunities to explore potential diagnostic markers differences in women with VVD, which may lead to targeted therapy.

Establishment of adequate levels of sialylation is crucial for sperm survival and function after insemination; however, the mechanism for the addition of the sperm sialome has not been identified. Here, we report evidence for several different mechanisms that contribute to the establishment of the mature sperm sialome. Directly quantifying the source of the nucleotide sugar CMP-beta-N-acetylneuraminic acid in epididymal fluid indicates that transsialylation occurs in the upper epididymis. Western blots for the low-molecular-mass sialoglycoprotein (around 20–50 kDa) in C57BL/6 mice epididymal fluid reflect that additional sialome could be obtained by glycosylphosphatidylinositol-anchored sialoglycopeptide incorporation during epididymal transit in the caput of the epididymis. Additionally, we found that in Cmah (CMP-N-acetylneuraminic acid hydroxylase)−/− transgenic mice, epididymal sperm obtained sialylated-CD52 from seminal vesicle fluid (SVF). Finally, we used Gfp (green fluorescent protein)+/+ mouse sperm to test the role of sialylation on sperm for protection from female leukocyte attack. There is very low phagocytosis of the epididymal sperm when compared to that of sperm coincubated with SVF. Treating sperm with Arthrobacter ureafaciens sialidase (AUS) increased phagocytosis even further. Our results highlight the different mechanisms of increasing sialylation, which lead to the formation of the mature sperm sialome, as well as reveal the sialome's function in sperm survival within the female genital tract.

Influenza A viruses (IAVs) utilize sialylated host glycans as ligands for binding and infection. The glycan-binding preference of IAV hemagglutinin (HA) is an important determinant of host specificity. Propagation of IAV in embryonated chicken eggs and cultured mammalian cells yields viruses with amino acid substitutions in the HA that can alter the binding specificity. Therefore, it is important to determine the binding specificity of IAV directly in primary samples since it reflects the actual tropism of virus in nature. We developed a novel platform for analysis of IAV binding specificity in samples that contain very low virus titers. This platform consists of a high-density flexible glycan display on magnetic beads, which promotes multivalent interactions with the viral HA. Glycan-bound virus is detected by quantifying the viral neuraminidase activity via a fluorogenic reporter, 2'-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. This method eliminates the need for labeling the virus and significantly enhances the sensitivity of detection.

Background: Mammalian sperm lose sialic acids during capacitation through unknown mechanisms. Results: Sialidases Neu1 and Neu3 are present on sperm. Their activity is required for capacitation and zona pellucida binding. Conclusion: Sperm sialidases modulate sperm surface sialic acids en route to fertilization. Significance: Understanding the mechanism of deciduous sialylation in sperm provides novel insights into sperm function and glycan-mediated fertility., Sialic acids (Sias) mediate many biological functions, including molecular recognition during development, immune response, and fertilization. A Sia-rich glycocalyx coats the surface of sperm, allowing them to survive as allogeneic cells in the female reproductive tract despite female immunity. During capacitation, sperm lose a fraction of their Sias. We quantified shed Sia monosaccharides released from capacitated sperm and measured sperm sialidase activity. We report the presence of two sialidases (neuraminidases Neu1 and Neu3) on mammalian sperm. These are themselves shed from sperm during capacitation. Inhibiting sialidase activity interferes with sperm binding to the zona pellucida of the ovum. A survey of human sperm samples for the presence of sialidases NEU1 and NEU3 identified a lack of one or both sialidases in sperm of some male idiopathic infertility cases. The results contribute new insights into the dynamic remodeling of the sperm glycocalyx prior to fertilization.

Background The ratio of Helicobacter pylori/NSAID-negative gastric ulcers is increasing. Idiopathic gastric ulcers have unique clinical and endoscopic features, and are associated with more bleeding complications and a higher mortality. Alterations in gastric mucin expression and sialylation pattern may be important in ulcer pathogenesis.

Aging is a multifactorial process that includes the lifelong accumulation of molecular damage, leading to age-related frailty, disability and disease, and eventually death. In this study, we report evidence of a significant correlation between the number of genes encoding the immunomodulatory CD33-related sialic acid-binding immunoglobulin-like receptors (CD33rSiglecs) and maximum lifespan in mammals. In keeping with this, we show that mice lacking Siglec-E, the main member of the CD33rSiglec family, exhibit reduced survival. Removal of Siglec-E causes the development of exaggerated signs of aging at the molecular, structural, and cognitive level. We found that accelerated aging was related both to an unbalanced ROS metabolism, and to a secondary impairment in detoxification of reactive molecules, ultimately leading to increased damage to cellular DNA, proteins, and lipids. Taken together, our data suggest that CD33rSiglecs co-evolved in mammals to achieve a better management of oxidative stress during inflammation, which in turn reduces molecular damage and extends lifespan.

MHC class I molecules play an essential role in the immune defense against intracellular infections. The hallmark of the MHC is its extensive degree of polymorphism at the population level. However, the present comparison of MHC class I gene intron variation revealed that chimpanzees have experienced a severe repertoire reduction at the orthologues of theHLA-A,-B, and-Cloci. The loss of variability predates the (sub)speciation of chimpanzees and did not effect other known gene systems. Therefore the selective sweep in the MHC class I gene may have resulted from a widespread viral infection. Based on the present results and the fact that chimpanzees have a natural resistance to the development of AIDS, we hypothesize that the selective sweep was caused by the chimpanzee-derived simian immunodeficiency virus (SIVcpz), the closest relative of HIV-1, or a closely related retrovirus. Hence, the contemporary chimpanzee populations represent the offspring of AIDS-resistant animals, the survivors of a HIV-like pandemic that took place in the distant past.

To determine the expression of membrane-bound mucins and glycan side chain sialic acids in Helicobacter pylori (H. pylori)-associated, non-steroidal inflammatory drug (NSAID)-associated and idiopathic-gastric ulcers.We studied a cohort of randomly selected patients with H. pylori (group 1, n = 30), NSAID (group 2, n = 18), combined H. pylori and NSAID associated gastric ulcers (group 3, n = 24), and patients with idiopathic gastric ulcers (group 4, n = 20). Immunohistochemistry for MUC1, MUC4, MUC17, and staining for Erythrina cristagalli agglutinin and Sambucus nigra agglutinin (SNA) lectins was performed on sections from the ulcer margins.Staining intensity of MUC17 was higher in H. pylori ulcers (group 1) than in idiopathic ulcers (group 4), 11.05 ± 3.67 vs 6.93 ± 4.00 for foveola cells, and 10.29 ± 4.67 vs 8.00 ± 3.48 for gland cells, respectively (P0.0001). In contrast, MUC1 expression was higher in group 4 compared group 1, 9.89 ± 4.17 vs 2.93 ± 5.13 in foveola cells and 7.63 ± 4.60 vs 2.57± 4.50 for glands, respectively (P0.0001). SNA lectin staining was increased in group 4, in parallel to elevated MUC1 expression, indicating more abundant α2-6 sialylation in that group.Cytoplasmic MUC17 staining was significantly decreased in the cases with idiopathic ulcer. The opposite was observed for both MUC1 and SNA lectin. This observation may reflect important pathogenic mechanisms, since different mucins with altered sialylation patterns may differ in their protection efficiency against acid and pepsin.

The surface antigen gene region from five chronic hepatitis B virus (HBV) infected chimpanzees was amplified by PCR and the sequence determined. Sequence comparison confirmed that all of the sequences were chimpanzee hepatitis B virus (chHBV) and they appeared to represent three distinct clusters or branches. To address the question of whether the three branches represented recently identified subspecies of chimpanzees, we determined the sequence of the mitochondrial DNA hypervariable D loop from hair samples obtained from these five chimpanzees. The results indicated that the three chHBV branches reflected three distinct subspecies of chimpanzees that are from different geographic regions in West Africa. The complete HBV sequence from members of the Pan troglodytes troglodytes cluster and the Pan troglodytes verus cluster are in the published literature; we determined the complete genome sequence for the third branch of HBV present in Pan troglodytes vellerosus.

The isolation of phylogenetically distinct primate immunodeficiency viruses from at least seven wild–born, captive chimpanzees indicates that viruses closely related to HIV–1 may be endemic in some wild chimpanzee populations. The search for the chimpanzee population or populations harbouring these viruses is therefore on. This paper attempts to answer the question of whether or not such populations of chimpanzees are likely to exist at all, and, if so, where they are likely to be found. We summarize what is known about gene flow in wild populations of chimpanzees, both between major phylogeographical subdivisions of the species, and within these subdivisions. Our analysis indicates that hitherto undocumented reproductively isolated chimpanzee populations may in fact exist. This conclusion is based on the observation that, despite limited geographical sampling and limited numbers of genetic loci, conventional notions of the nature and extent of chimpanzee gene flow have recently been substantially revised. Molecular genetic studies using mitochondrial DNA sequences and hypervariable nuclear microsatellite markers have indicated the existence of heretofore undocumented barriers to chimpanzee gene flow. These studies have identified at least one population of chimpanzees genetically distinct enough to be classified into a new subspecies ( Pan troglodytes vellerosus ). At the same time, they have called into question the long–accepted genetic distinction between eastern chimpanzees ( Pan troglodytes schweinfurthii ) and western equatorial chimpanzees ( Pan troglodytes troglodytes ). The same studies have further indicated that gene flow between local populations is more extensive than was previously thought, and follows patterns sometimes inconsistent with those documented through direct behavioural observation. Given the apparently incomplete nature of the current understanding of chimpanzee gene flow in equatorial Africa, it seems reasonable to speculate that a chimpanzee population or populations may exist which both harbour the putative HIV–1 ancestor, and which have remained reproductively isolated from other chimpanzee populations over the time–scale relevant to the evolution of the SIVcpz–HIV–1 complex of viruses. Continued extensive sampling of wild chimpanzee populations, both for their genes and their viruses, should be performed quickly considering the high probability of extinction that many wild chimpanzee populations face today. The history of human–chimpanzee contacts is discussed.

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight(1-3). Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract(3,4). In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins(5-9). The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms(8,10-12). As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media. Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods(13). Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo. In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods. We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Sialic acids are a diverse family of monosaccharides widely expressed on all cell surfaces of vertebrates and so-called “higher” invertebrates, and on certain bacteria that interact with vertebrates. This overview surveys examples of biological roles of sialic acids in immunity, with emphasis on an evolutionary perspective. Given the breadth of the subject, the treatment of individual topics is brief. Subjects discussed include biophysical effects regulation of factor H; modulation of leukocyte trafficking via selectins; Siglecs in immune cell activation; sialic acids as ligands for microbes; impact of microbial and endogenous sialidases on immune cell responses; pathogen molecular mimicry of host sialic acids; Siglec recognition of sialylated pathogens; bacteriophage recognition of microbial sialic acids; polysialic acid modulation of immune cells; sialic acids as pathogen decoys or biological masks; modulation of immunity by sialic acid O-acetylation, sialic acids as antigens and xeno-autoantigens; anti-sialoglycan antibodies in reproductive incompatibility, and sialic-acid based blood groups.

A comparative analysis of bacterial and mammalian glycomes based on the statistical analysis of two major carbohydrate databases, Bacterial Carbohydrate Structure Data Base (BCSDB) and GLYCOSCIENCES.de (GS), is presented. An in-depth comparison of these two glycomes reveals both striking differences and unexpected similarities. Within the prokaryotic kingdom, we focus on the glycomes of seven classes of pathogenic bacteria with respect to (i) their most abundant monosaccharide units; (ii) disaccharide pairs; (iii) carbohydrate modifications; (iv) occurrence and use of sialic acids; and (v) class-specific monosaccharides. The aim of this work is to gain insights into unique carbohydrate patterns in bacteria. Data interpretation reveals significant trends in the composition of specific carbohydrate classes as result of evolution-driven structural adaptations of bacterial pathogens and symbionts to their mammalian hosts. The differences are discussed in light of their value for biomedical applications, such as the targeting of unique glycosyl transferases, vaccine development, and devising novel diagnostic tools. peerReviewed

We report here that human T cells give much stronger proliferative responses to specific activation via the T cell receptor (TCR) than those from chimpanzees, our closest evolutionary relatives. Nonspecific activation using phytohemagglutinin was robust in chimpanzee T cells, indicating that the much lower response to TCR simulation is not due to any intrinsic inability to respond to an activating stimulus. CD33-related Siglecs are inhibitory signaling molecules expressed on most immune cells and are thought to down-regulate cellular activation pathways via cytosolic immunoreceptor tyrosine-based inhibitory motifs. Among human immune cells, T lymphocytes are a striking exception, expressing little to none of these molecules. In stark contrast, we find that T lymphocytes from chimpanzees as well as the other closely related “great apes” (bonobos, gorillas, and orangutans) express several CD33-related Siglecs on their surfaces. Thus, human-specific loss of T cell Siglec expression occurred after our last common ancestor with great apes, potentially resulting in an evolutionary difference with regard to inhibitory signaling. We confirmed this by studying Siglec-5, which is prominently expressed on chimpanzee lymphocytes, including CD4 T cells. Ab-mediated clearance of Siglec-5 from chimpanzee T cells enhanced TCR-mediated activation. Conversely, primary human T cells and Jurkat cells transfected with Siglec-5 become less responsive; i.e., they behave more like chimpanzee T cells. This human-specific loss of T cell Siglec expression associated with T cell hyperactivity may help explain the strikingly disparate prevalence and severity of T cell-mediated diseases such as AIDS and chronic active hepatitis between humans and chimpanzees.

Efforts to control the global pandemic of human hepatitis B virus (hHBV) infection have been hampered by incomplete understanding of viral–host interactions in this disease. This situation has been confounded by the fact that hHBV has a limited host range and cannot be propagated in simple cell culture (1). Reproducible experimental infection with determination of infectivity was demonstrated in chimpanzees (Pan troglodytes), but not other primates (2–4), long before other animal models such as the woodchuck were identified. After successful inoculation of chimpanzees was reported in 1972, multiple institutions, including a multigroup collaboration between the FDA, CDC, and NIH, initiated studies to evaluate them as a model for the study of HBV. For the majority of studies only chimpanzees “with no prior exposure” to virus were used because those with positive serology [either from exposure to hHBV or chimpanzee HBV (chHBV)], with an estimated prevalence of 3–6% in Africa (5), were not reproducibly susceptible to infection (3). It has been widely reported that the effects of HBV infection in chimpanzees are milder than in humans, that is, few have developed fulminant hepatitis, and inoculated chimpanzees exhibit few symptoms or signs of infection. Furthermore, the incidence of chronic infection with HBV (see Table 2) and horizontal and vertical transmission (from mother to offspring) in chimpanzees is lower than in humans (6,7). Chimpanzees have been the cornerstone of all research on infectivity of HBV and safety and efficacy of vaccines. Chronic HBV infection occurs in approx 5% of experimentally infected chimpanzees, defined as persistence of hepatitis B surface antigen (HBsAg) for 12 months (approx 2⁄3 clear HBsAg between 6 and 12 months of infection) (8). Liver biopsies of chimpanzees with chronic HBsAg have never been reported to have more severe abnormalities than mild persistent hepatitis (8–11), but we were unable to find published

Many microbial pathogens and toxins recognize animal cells via cell surface sialic acids (Sias) that are alpha 2-3- or alpha 2-8-linked to the underlying glycan chain. Human influenza A/B viruses are unusual in preferring alpha 2-6-linked Sias, undergoing a switch from alpha 2-3 linkage preference during adaptation from animals to humans. This correlates with the expression of alpha 2-6-linked Sias on ciliated human airway epithelial target cells and of alpha 2-3-linked Sias on secreted soluble airway mucins, which are unable to inhibit virus binding. Given several known differences in Sia biology between humans and apes, we asked whether this pattern of airway epithelial Sia linkages is also human-specific. Indeed, we show that since the last common ancestor with apes, humans underwent a concerted bidirectional switch in alpha 2-6-linked Sia expression between airway epithelial cell surfaces and secreted mucins. This can explain why the chimpanzee appears relatively resistant to experimental infection with human Influenza viruses. Other tissues showed additional examples of human-specific increases or decreases in alpha 2-6-linked Sia expression and only one example of a change specific to certain great apes. Furthermore, while human and great ape leukocytes both express alpha 2-6-linked Sias, only human erythrocytes have markedly up-regulated expression. These cell type-specific changes in alpha 2-6-Sia expression during human evolution represent another example of a human-specific change in Sia biology. Because the data set involves multiple great apes, we can also conclude that Sia linkage expression patterns can be conserved during millions of years of evolution within some vertebrate taxa while undergoing sudden major changes in other closely related ones.

Most blood plasma proteins are glycosy- lated. These glycoproteins typically carry sialic acid-bear- ing sugar chains, which can modify the observed molecu- lar weights and isoelectric points of those proteins during electrophoretic analyses. To explore changes in protein expression and glycosylation that occurred during great ape and human evolution, we subjected multiple blood plasma samples from all these species to high-resolution proteomic analysis. We found very few species-specific differences, indicating a remarkable degree of conserva- tion of plasma protein expression and glycosylation during ;12 million years of evolution. A few lineage-specific dif- ferences in protein migration were noted among the great apes. The only obvious differences between humans and all great apes were an apparent decrease in transthyretin (prealbumin) and a change in haptoglobin isoforms (the latter was predictable from prior genetic studies). Quan- titative studies of transthyretin in samples of blood plasma (synthesized primarily by the liver) and of cere- brospinal fluid (synthesized locally by the choroid plexus of the brain) confirmed ;2-fold higher levels in chimpan- zees compared to humans. Since transthyretin binds thy- roid hormones, we next compared plasma thyroid hor- mone parameters between humans and chimpanzees. The results indicate significant differences in the status of thyroid hormone metabolism, which represent the first known endocrine difference between these species. Nota- bly, thyroid hormones are known to play major roles in the development, differentiation, and metabolism of many or- gans and tissues, including the brain and the cranium. Also, transthyretin is known to be the major carrier of thyroid hormone in the cerebrospinal fluid, likely regulat- ing delivery of this hormone to the brain. A potential secondary difference in retinoid (vitamin A) metabolism is also noted. The implications of these findings for explain- ing unique features of human evolution are discussed. Am J Phys Anthropol 115:99 -109, 2001. © 2001 Wiley-Liss, Inc.

The remarkable similarity among the genomes of humans and the African great apes could warrant their classification together as a single genus. However, whereas there are many similarities in the biology, life history, and behavior of humans and great apes, there are also many striking differences that need to be explained. The complete sequencing of the human genome creates an opportunity to ask which genes are involved in those differences. A logical approach would be to use the chimpanzee genome for comparison and the other great ape genomes for confirmation. Until such a great ape genome project can become reality, the next best approach must be educated guesses of where the genetic differences may lie and a careful analysis of differences that we do know about. Our group recently discovered a human-specific inactivating mutation in the CMP-sialic acid hydroxylase gene, which results in the loss of expression of a common mammalian cell-surface sugar throughout all cells in the human body. We are currently investigating the implications of this difference for a variety of issues relevant to humans, ranging from pathogen susceptibility to brain development. Evaluating the uniqueness of this finding has also led us to explore the existing literature on the broader issue of genetic differences between humans and great apes. The aim of this brief review is to consider a listing of currently known genetic differences between humans and great apes and to suggest avenues for future research. The differences reported between human and great ape genomes include cytogenetic differences, differences in the type and number of repetitive genomic DNA and transposable elements, abundance and distribution of endogenous retroviruses, the presence and extent of allelic polymorphisms, specific gene inactivation events, gene sequence differences, gene duplications, single nucleotide polymorphisms, gene expression differences, and messenger RNA splicing variations. Evaluation of the reported findings in all these categories indicates that the CMP-sialic hydroxylase mutation is the only one that has so far been shown to result in a global biochemical and structural difference between humans and great apes. Several of the other known genetic dissimilarities deserve more exploration at the functional level. Among the areas of focus for the future should be genes affecting development, mental maturation, reproductive biology, and other aspects of life history. The approaches taken should include both going from the genome up to the adaptive potential of the organisms and going from novel adaptive regimes down to the relevant repercussions in the genome. Also, as much as we desire a simple genetic explanation for the human phenomenon, it is much more probable that our evolution occurred in multiple genetic steps, many of which must have left detectable footprints in our genomes. Ultimately, we need to know the exact number of genetic steps, the order in which they occurred, and the temporal, spatial, environmental, and cultural contexts that determined their impact on human evolution.

Malaria caused by Plasmodium falciparum (“malignant malaria”) is one of the most devastating pathogens of humans (1). Plasmodium reichenowi, which infects chimpanzees and gorillas, is the closest relative of P. falciparum (2). In early 20th-century experiments (never to be replicated), blood from P. reichenowi-infected chimpanzees was injected into humans, but failed to produce infections (3). Conversely, chimpanzees injected with P.falciparum-infected human blood suffered no in fection. Taken together, these data suggested that each parasite had coevolved with its host, but did not rule out chimpanzee to human transmission, or vice versa. In this issue of PNAS, Rich et al. (4) provide an answer to this malignant malaria “mystery” and confirm a prediction that we and our colleagues made earlier (5).

We are very glad to read of Dr. Previc’s enthusiasm for our recent findings on thyroid hormone differences between humans and great apes (Gagneux et al., 2001). We are responding herewith to some of the interesting issues that he has raised. Our paper presented human-ape comparative results on plasma thyroid hormone levels and levels of transthyretin (TTR) in plasma and cerebrospinal fluid (CSF). Given the crucial role of thyroid hormone in brain development, we suggested that developmental studies of humans and great ape thyroid hormone levels are warranted, especially for the CSF/brain side of the blood brain barrier. We did not hypothesize about how such hormonal differences originally came about. Extrapolating from currentday human-ape differences to the series of events that brought about these changes sometime during our evolutionary past is fraught with difficulty. However, we are glad if our results provide potential support to other existing hypotheses. Increased consumption of animal foods and frequent strenuous exercise may indeed have played a crucial role in resetting dopaminergic levels in hominoids, as proposed by Dr. Previc’s extensively researched paper on this subject (Previc, 1999). However, it is also evident that present-day humans can achieve aboveaverage cognitive capacities in the absence of meatrich diets or increased physical activity levels. As far