Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

Late diagnosis of Cushing's disease in a child: what lessons can be learned?

We present a case of late diagnosis of an adolescent female with Cushing's disease or excess glucocorticoid secretion due to the presence of an adrenocorticotropic hormone (ACTH) secreting tumor on the pituitary. Her main complaints at first presentation in 2008 were short stature and weight gain. Although these are the main criteria for testing for Cushing's syndrome outlined by the 2008 Endocrine Society guidelines, they were not enough to lead to a timely diagnosis. The reasons may include the endocrinologists believing that additional more common symptoms needed to be present to test for Cushing's syndrome. Consequently, the guidelines were not followed leading to placing the child on growth hormone (GH) with no further evaluation until well after 1 year of minimal growth. While Cushing's syndrome is rare in children and hard to diagnose, a more collaborative effort among pediatricians, endocrinologists, radiologists, surgeons, as well as parents, is required to diagnose and help cure children with this disease.

A seminal monograph: Mackay and Burnet's Autoimmune diseases.

Mackay and Burnet's Autoimmune diseases, published in 1962, marked the beginning of autoimmunity as a clinical science and led to the future acceptance of the existence of autoimmunity. While there is still controversy regarding the mechanisms of autoimmunity, the authors' insightful hypothesis based on clonal selection theory and the emergence of "forbidden clones", due to somatic mutations, is still current, with recent evidence giving further credence to this hypothesis. We salute Mackay and Burnet on the 50th anniversary of this seminal publication. It is particularly pleasing that it has an iconic Australian origin.

Bladder dysfunction in multiple sclerosis.

Multiple sclerosis (MS) is a relatively common disease of young adults. Patients with MS can have a wide range of symptoms and may develop significant disability. The cause of MS is unknown, but immunological mechanisms are important. In MS, the pathological features include prominent demyelination and inflammation, but there is also evidence of neurodegeneration. Bladder symptoms are common in MS. The bladder is under neural control, and bladder disturbance is usually attributed to demyelination or loss of axons from the neural pathways, particularly those in the spinal cord, that control the bladder. However, as with other symptoms in MS, the presence of bladder disturbance does not always correlate well with MRI lesions. We speculate that other possible causes of bladder dysfunction in MS might include the effects of circulating toxic factors. Urgency of micturition is prominent in MS, and better understanding of the receptors involved in bladder sensation suggests possible treatment strategies through inhibiting these receptors.

Autoantibody-mediated bowel and bladder dysfunction in a patient with chronic, nondiabetic neuropathy.

Physiological techniques can be used to detect novel autoantibodies causing alteration of autonomic function after passive transfer to mice. Previously, such antibodies have been detected in patients with type I diabetes mellitus, myasthenia gravis, and Sjogren's syndrome. We now describe a patient with an idiopathic nondiabetic neuropathy with prominent autonomic symptoms, including bladder and bowel dysfunction. Physiological assays of whole colon and bladder were used to determine the presence in the patient serum of functional autoantibodies capable of mediating autonomic dysfunction. Immunoglobulin G (IgG) from this patient was able to disrupt bladder and bowel function on passive transfer to mice. This is a new pattern of autoantibody-mediated abnormality. Although the target antigen is unknown, it is likely to be a cell-surface receptor or ion channel. This case highlights the usefulness of passive transfer studies in detecting functional antibodies in patients with autonomic neuropathy.

A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. III. Comparative performance characteristics of academic and manufacturers' laboratories.

OBJECTIVE: To analyze the performance of different commercial enzyme immunoassay (EIA) kits for measuring antinuclear antibodies (ANA) specific for dsDNA, SSB/La, Sm, and Scl-70. METHODS: EIA kits for detection of ANA from 9 commercial manufacturers were evaluated. The manufacturers were advised that they would be sent coded sera containing mixtures of the Arthritis Foundation/Centers for Disease Control reference reagents, and that they were to use their own test kits to analyze the antibody specificities of these sera and to report the data, in optical density (OD) units or their equivalent. Independently, 12 investigators in academic institutions who have done research in this field agreed to participate in a parallel study. The concentration of the antibodies and the specificities were blinded to the analysts and the coefficients of variation (CV) were computed for each participant. RESULTS: There were statistically significant differences between laboratories in terms of CV for all 9 kits tested. With the exception of one kit, there were no significant CV differences between the various autoantibody kits provided by each manufacturer and, with the exception of kits from 2 manufacturers, there were no significant differences between the various antibody kits in terms of reproducibility (CV). From the point of view of interlaboratory variability, manufacturers could be separated into either a high or low performance group. CONCLUSION: We found a disconcertingly large range of performance characteristics in the various laboratories, which could be quite detrimental in routine utilization of EIA ANA kits. Clinicians should be aware of the performance issues raised in our study, and should know and be involved in how their service laboratory assesses its own performance and the performance of commercial testing systems utilized. Manufacturers and clinical laboratories need to exercise constant quality assurance and surveillance of kit performance in the hands of medical laboratory technologists involved in routine testing.

Tolerance through indifference: autoreactive B cells to the nuclear antigen La show no evidence of tolerance in a transgenic model.

Systemic autoimmune diseases are characterized by the production of high titer autoantibodies specific for ubiquitous nuclear self-Ags such as DNA, Sm, and La (SS-B), so the normal mechanisms of B cell tolerance to disease-associated nuclear Ags have been of great interest. Mechanisms of B cell tolerance include deletion, anergy, developmental arrest, receptor editing, and B cell differentiation to the B-1 subtype. However, recent studies in our laboratory have suggested that B cell tolerance to the nuclear autoantigen La is limited in normal mice, and tolerance may reside primarily in the T cell compartment. To test this hypothesis, we created Ig transgenic mice expressing the IgM H chain from an mAb specific for a xenogeneic epitope within human La (hLa). These mice were bred with hLa-transgenic mice that constitutively express hLa in a manner comparable to endogenous mouse La. Between 5-15% of transgenic B cells developing in the absence of hLa were specific for hLa, and these cells were neither depleted nor developmentally arrested in the presence of endogenous hLa expression. Instead, these autoreactive B cells matured normally and differentiated into Ab-forming cells, capable of secreting high titer autoantibody. Additionally, the life span of autoreactive hLa-specific B cells was not reduced, and they were phenotypically and functionally indistinguishable from naive nonautoreactive hLa-specific B cells developing in the absence of hLa. Together these data suggest a lack of intrinsic B cell tolerance involving any known mechanisms indicating that these autoreactive B cells are indifferent to their autoantigen.

Immunomodulatory effects of estrogen and progesterone replacement in a nonhuman primate model.

A novel postmenopausal nonhuman primate model consisting of healthy young and old ovariectomized rhesus macaques was used to assess the short-term immunomodulatory effects of transdermally administered estrogen and progesterone. Specifically, we determined estrogen- and progesterone-induced changes in absolute numbers of circulating lymphocytes (B lymphocytes, CD4+ lymphocytes, and CD8+ lymphocytes) as well as lymphocytes expressing the activation markers CD25 and CD69. In addition, we assessed B and T lymphocyte activity, i.e, immunoglobulin (Ig) and interferon-gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMCs). In general, treatment with estrogen or progesterone resulted in decreased lymphocyte numbers and in down-modulation of activation markers. In addition, hormone replacement resulted in a decreasing trend for PBMC IFN-gamma production, whereas PBMC Ig production was minimally affected. Hormone treatment seemed to influence young and old animals differently, with the young animals appearing more susceptible to its immune system-related effects. These results indicate that, in our animal model exogenously administered hormones may dynamically interact with the immune system, resulting in in vivo modulation of lymphocyte numbers and activity.

Reproductive parameters in the sooty mangabey (Cercocebus torquatus atys).

Data obtained over a 7.5 year period from a captive group of sooty mangabeys were analyzed to examine the relationship between demographic variables and reproductive events. Perineal swelling and reproductive data collected from 67 females contributed to the analyses. Sexual maturation, as marked by the onset of swelling cycles, was recorded at an average age of 36.4 ± 1.1 (mean ± SEM) months and first parturition at 56.5 ± 1.8 months, indicating a period of adolescent sterility of over 1 year. A total of 198 births were recorded, of which 165 (83.3%) were live births with infant survival to 30 days of age. Both parity and the duration of infant survival influenced interbirth interval, with the shortest interval observed in multiparous females with infants surviving less than 6 months (10.0 ± 0.5 months) and the longest in primiparous females with infants surviving more than 12 months (22.4 ± 1.2 months). Infant survival did not vary significantly according to the age class of the mother. Births and the onset of perineal swellings in young females showed a seasonal distribution, with swelling cycles and conceptions rarely occurring from April through September.

Survival rate and life span of rhesus monkeys at the Yerkes regional primate research center.

This paper describes the survival rates of 763 rhesus monkeys maintained at the Yerkes Regional Primate Research Center (YRPRC). The survival rates were determined by methods used to calculate survival rates of human populations. The monkeys were divided into 3 groups based on their specific life histories. Group I monkeys were wild-born and were housed singly from the time they came into captivity at about 2 years of age. Group II monkeys were born either in the wild or in captivity and were housed in social groups since their acquisition at ages 2 to 8 years. Group III monkeys were born at the YRPRC and housed in social groups. Due to these differences in life histories, direct comparisons among survival curves of the 3 groups are, at best, tenuous, as are comparisons with populations maintained at other facilities. In the present study the highest mortality rate occurred during the first month of life. The maximum life span attained in our group I was 35 years, with only 6.2% of monkeys in this group attaining an age beyond 30 years.

Wounding patterns in three species of captive macaques.

The incidence of wounding in captive groups of rhesus (Macaca mulatta), pigtail (M. nemestrina), and stumptail (M. arctoides) macaques was studied for 21 months. Groups were monitored daily for evidence of wounding. Wounded animals were captured, treated by veterinary staff, and returned following recovery. Records were kept on the age, sex, and species of the recipient, along with the type and location of wound. In each species of macaque, adult males incurred the highest frequency of wounds and multiple wounds of any age-sex class. This contrasted with previously reported behavioral data indicating low frequencies of aggression received by adult males, especially contact aggression and bites. These discrepancies indicate wounding frequencies do not necessarily correspond with behavioral measures of aggression. Inhibition of aggression directed toward infants and the selective avoidance of bites directed to vital body regions were presented as possible mechanisms that modify intragroup aggression. Increased wounding in the birth season under captive conditions suggests that the pattern of increased wounding reported during the breeding season under freeranging conditions may reflect xenophobic responses to immigrating males, rather than direct male-male competition for estrous females.

Infant carrying by adult male mangabeys (Cercocebus atys).

Sixty-five interactions where an adult male carried an infant in the proximity of a second male were observed during a 19-mo period in a captive sooty mangabey (Cercocebus atys) social group. The behavior was distributed nonrandomly in both the adult male and infant classes. In all but three of the interactions, the recently deposed alpha male carried an infant in the presence of the newly ascendant dominant male. In the first phase of the study, infants that were carried included the entire unweaned infant cohort (n = 5) born before the rank reversal. The rate of carrying in this class declined as a function of increasing infant age and time since the rank reversal. Infant carrying was not observed in the context of fighting, which was rare, and intermale aggression never preceded the behavior. However, in 40% of the cases, carrying occurred after an infant had been threatened by the dominant male. These results suggest that infant carrying served to protect the infant from aggression rather than to exploit the infant as an agonistic buffer. The data did not unequivocally support the postulate that carrying may be a form of paternal care since paternity could not be assessed. The similar structural and contextual patterns of infant carrying in this species suggest a common origin for triadic male-infant interactions in mangabeys and baboons.

Effects of age, lactation, and repeated cycles on rhesus monkey copulatory intervals.

Young, sexually mature female rhesus monkeys copulate on more days prior to conception than do older females, and this prolonged discrete mating period is associated with an earlier rise in serum estradiol prior to the first ovulation of the breeding season. The influence of repeated ovulatory cycles and the presence of a suckling infant on the copulatory patterns were examined in two separate analyses. Extending previous work, young, nulliparous females copulated on more days at the first ovulation of the breeding season than did older, multiparous females. However, the duration of the copulatory period at the second ovulation of the breeding season was similar and significantly shorter for both age groups. Furthermore, the presence of a suckling infant did not influence the duration of the mating periods in adult, multiparous females. The onset of copulatory behavior for all females was associated with serum estradiol concentrations of approximately 90 pg/ml, indicating that the age and cycle differences in the duration of the copulatory periods are due to the time course of serum estradiol prior to ovulation. A separate, longitudinal analysis of the duration of the mating period associated with the first ovulation of three successive breeding seasons indicated that females copulated on more days during their first ovulatory cycle of their first breeding season. These data indicate that the copulatory interval is longer for females during the first ovulation of the breeding season, and this pattern is accentuated in young, sexually mature animals.

Attacks on neonates by a male mangabey (Cercocebus atys).

Aggressive behavior by an adult male toward selected infants and their mothers was observed during a long-term study of reproduction in a captive social group of sooty mangabeys (Cercocebus atys). The highest-ranking adult male in this group was observed to attack and bite three neonates out of a total of 13 infants born in 1982. All three attacks were directed attacks in which infants were grabbed from their mothers and bitten in the head. The first infant was fatally injured; the other two probably would have sustained fatal wounds had the male's canines not been blunted beforehand and had observers not intervened. The attacks were preceded by a pattern in which the male persistently stalked or chased the mother-infant pairs, a behavior first observed in the hours immediately following parturition. Unlike attacks in wild baboon groups following male immigration, these attacks on infants occurred in a stable social group in which the male attacker had been a lifelong resident. This male, however, had gained alpha rank only 3 months before attacking the first infant. These attacks, in the context of other evidence of aggression and wounding, are discussed relative to current models of infanticide in primates.

Female mating relationships in rhesus monkeys.

Descriptions of the sexual behavior of social-living rhesus monkeys have indicated that mating patterns are characterized by the formation of consort pairs (mutually exclusive male-female associations) and also that females typically mate with multiple males. These reports raise the question of whether these are two different mating strategies or merely sequential phases in a unitary mating pattern. In order to examine this issue, the copulatory behavior of 27 adult females living in a heterosexual group housed in an outdoor compound was monitored both by group scan and focal sampling technique. Females displayed discrete periods of mating, characterized by one or more copulations per 4 hr observation period. During their sexually active periods, females copulated with multiple males (X̄ = 5.22, SE = 0.37). Among the 103 cases where females had multiple copulatory series on the same day, the successive series involved new males significantly more often than the previous partner. When a female did copulate repeatedly with a male on a given day, both she and her partner were significantly more often of high social rank. Furthermore, the interval between successive copulations was significantly longer when the second copulation was with her previous partner as compared to a different male. Analysis of female behavior for a one-hour period following a copulation indicated that females continued to be sexually active and that those new sexual interactions more often involved a new male. Thus, females not only copulated with multiple males throughout their inclusive period of sexual activity, but also interacted with multiple males on any give day they were sexually active. Since repeated copulations with the same male were rare and occurred when both members of the pair were high ranking, the consort relationship probably does not describe a characteristic mating strategy. The more pervasive pattern is for a female to sexually interact and have copulations with multiple males.

The effectiveness of sampling methods in detecting copulatory behavior in Macaca arctoides.

The copulatory behavior of a social group of 18 stumptail macaques was observed for 171 hours over a period of 5 months. The occurrence of copulation and several quantitative measures of copulation were compared using sampling periods of 2 hours daily and continuous dawn-to-dusk observation. In this environment copulation was found to occur in brief bouts of relatively short duration. Copulation was detected on 34% of the days observed with 2 hr sampling and 100% of the days with DTD sampling. Thus, brief limited-time samples of behavior are not adequate for reliably detecting copulation in this species under social group-living conditions.