Increased photic sensitivity for phase resetting but not melatonin suppression in Siberian hamsters under short photoperiods.

Light regulates a variety of behavioral and physiological processes, including activity rhythms and hormone secretory patterns. Seasonal changes in the proportion of light in a day (photoperiod) further modulate those functions. Recently, short (SP) versus long days (LP) were found to markedly increase light sensitivity for phase shifting in Syrian hamsters. To our knowledge, photoperiod effects on light sensitivity have not been studied in other rodents, nor is it known if they generalize to other circadian responses. We tested whether photic phase shifting and melatonin suppression vary in Siberian hamsters maintained under LP or SP. Select irradiances of light were administered, and shifts in activity were determined. Photic sensitivity for melatonin suppression was examined in a separate group of animals via pulses of light across a 4 log-unit photon density range, with post-pulse plasma melatonin levels determined via RIA. Phase shifting and melatonin suppression were greater at higher irradiances for both LP and SP. The lower irradiance condition was below threshold for phase shifts in LP but not SP. Melatonin suppression did not vary by photoperiod, and the half saturation constant for fitted sigmoid curves was similar under LP and SP. Thus, the photoperiodic modulation of light sensitivity for phase shifting is conserved across two hamster genera. The dissociation of photoperiod effects on photic phase shifting and melatonin suppression suggests that the modulation of sensitivity occurs downstream of the common retinal input pathway. Understanding the mechanistic basis for this plasticity may yield therapeutic targets for optimizing light therapy practices.

Failure of T-cell homeostasis preceding AIDS in HIV-1 infection. The Multicenter AIDS Cohort Study.

We and others have postulated that a constant number of T lymphocytes is normally maintained without regard to CD4+ or CD8+ phenotype ('blind' T-cell homeostasis). Here we confirm essentially constant T-cell levels (despite marked decline in CD4+ T cells and increase in CD8+ T cells) in homosexual men with incident human immunodeficiency virus, type 1 (HIV-1), infection who remained free of acquired immunodeficiency syndrome (AIDS) for up to eight years after seroconversion. In contrast, seroconverters who developed AIDS exhibited rapidly declining T cells (both CD4+ and CD8+) for approximately two years before AIDS, independent of the time between seroconversion and AIDS, suggesting that homeostasis failure is an important landmark in HIV disease progression. Given the high rate of T-cell turnover in HIV-1 infection, blind T-cell homeostasis may contribute to HIV pathogenesis through a CD8+ T lymphocytosis that interferes with regeneration of lost CD4+ T cells.

Familial aggregation of food allergy and sensitization to food allergens: a family-based study.

BACKGROUND: The increasing prevalence of food allergy (FA) is a growing clinical and public health problem. The contribution of genetic factors to FA remains largely unknown. OBJECTIVE: This study examined the pattern of familial aggregation and the degree to which genetic factors contribute to FA and sensitization to food allergens. METHODS: This study included 581 nuclear families (2,004 subjects) as part of an ongoing FA study in Chicago, IL, USA. FA was defined by a set of criteria including timing, clinical symptoms obtained via standardized questionnaire interview and corroborative specific IgE cut-offs for > or =95% positive predictive value (PPV) for food allergens measured by Phadia ImmunoCAP. Familial aggregation of FA as well as sensitization to food allergens was examined using generalized estimating equation (GEE) models, with adjustment for important covariates including age, gender, ethnicity and birth order. Heritability was estimated for food-specific IgE measurements. RESULTS: FA in the index child was a significant and independent predictor of FA in other siblings (OR=2.6, 95% CI: 1.2-5.6, P=0.01). There were significant and positive associations among family members (father-offspring, mother-offspring, index-other siblings) for total IgE and specific IgE to all the nine major food allergens tested in this sample (sesame, peanut, wheat, milk, egg white, soy, walnut, shrimp and cod fish). The estimated heritability of food-specific IgE ranged from 0.15 to 0.35 and was statistically significant for all the nine tested food allergens. CONCLUSION: This family-based study demonstrates strong familial aggregation of FA and sensitization to food allergens, especially, among siblings. The heritability estimates indicate that food-specific IgE is likely influenced by both genetic and environmental factors. Together, this study provides strong evidence that both host genetic susceptibility and environmental factors determine the complex trait of IgE-mediated FA.

Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women.

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.

Circadian waveform bifurcation, but not phase-shifting, leaves cued fear memory intact.

In mammals, memory acquisition and retrieval can be affected by time of day, as well as by manipulations of the light/dark cycle. Under bifurcation, a manipulation of circadian waveform, two subjective days and nights are experimentally induced in rodents. We examined the effect of bifurcation on Pavlovian fear conditioning, a prominent model of learning and memory. Here we demonstrate that bifurcation of the circadian waveform produces a small deficit in acquisition, but not on retrieval of fear memory. In contrast, repeated phase-shifting in a simulated jet-lag protocol impairs retrieval of memory for cued fear. The results have implications for those attempting to adjust to shift-work or other challenging schedules.

In synch but not in step: Circadian clock circuits regulating plasticity in daily rhythms.

The suprachiasmatic nucleus (SCN) is a network of neural oscillators that program daily rhythms in mammalian behavior and physiology. Over the last decade much has been learned about how SCN clock neurons coordinate together in time and space to form a cohesive population. Despite this insight, much remains unknown about how SCN neurons communicate with one another to produce emergent properties of the network. Here we review the current understanding of communication among SCN clock cells and highlight a collection of formal assays where changes in SCN interactions provide for plasticity in the waveform of circadian rhythms in behavior. Future studies that pair analytical behavioral assays with modern neuroscience techniques have the potential to provide deeper insight into SCN circuit mechanisms.

Daily novel wheel running reorganizes and splits hamster circadian activity rhythms.

The phenomenon of splitting of locomotor activity rhythms in constant light has implied that the mammalian circadian pacemaker is composed of multiple interacting circadian oscillators. Exposure of male Syrian hamsters to novel running wheels also induces splitting in some reports, although novel wheel running (NWR) is better known for its effects on altering circadian phase and the length of the free-running period. In three experiments, the authors confirm and extend earlier reports of split rhythms induced by NWR. Male Syrian hamsters, entrained to LD 14:10, were transferred for 6 to 11 consecutive days to darkened novel Wahmann wheels at ZT 4 and were returned to their home cages at ZT 9. All hamsters ran robustly in the novel wheels. NWR caused a marked reorganization of home cage wheel-running behavior: Activity onsets delayed progressively with each additional day of NWR. After 11 days, activity onset in the nighttime scotophase was delayed by 7 h and disappeared completely in 2 hamsters (Experiment 1). After 6 to 7 days of NWR (Experiment 2), activity onset delayed by 5 h. Transfer of hamsters to constant darkness (DD) after 7 days of NWR revealed clearly split activity rhythms: The delayed nighttime activity bout was clearly identifiable and characterized by a short duration. A second bout associated with the former time of NWR was equally distinct and exhibited a similarly short duration. These components rejoined after 3 to 5 days in DD accomplished via delays and advances of the nighttime and afternoon components, respectively. The final experiment established that rejoining of activity components could be prevented by perpetuating the light-dark:light-dark cycle used to induce split rhythms. The data suggest that NWR causes selective phase shifting of some circadian oscillators and that component oscillators interact strongly in constant darkness.

Photoperiodism in hamsters: abrupt versus gradual changes in day length differentially entrain morning and evening circadian oscillators.

In studies of photoperiodism, animals typically are transferred abruptly from a long (e.g., 16 h light per day [16L]) to a short (8L) photoperiod, and circadian oscillators that regulate pineal melatonin secretion are presumed to reentrain rapidly to the new photocycle. Among rats and Siberian hamsters, however, reentrainment rates vary depending on whether additional darkness is added to morning or evening, and a subset of hamsters (nonresponders) fails ever to reentrain normally to short photoperiods. The authors assessed whether several short-day responses occurred at different rates when darkness was extended into morning versus evening hours and the effectiveness of abrupt versus gradual shortening in day lengths (DLs). Entrainment patterns of photoresponsive hamsters also were compared to those of photononresponsive hamsters. Responsive hamsters transferred on a single day from 16L to 8L underwent more rapid gonadal regression, weight loss, decreases in follicle-stimulating hormone titers, and expansion of nocturnal locomotor activity when darkness was added to morning versus evening. When the dark phase was extended gradually by 8 h over 16 weeks, short-day responses occurred at the same rate whether darkness was appended to morning or evening or was added symmetrically. Darkness added to evening promoted more rapid short-day responses when it was added gradually rather than abruptly, despite the fact that average DLs were significantly shorter for the latter group. Among nonresponders, morning extensions of darkness transiently increased activity duration, whereas evening extensions did not. Gradual and abrupt decreases in DL differentially affect entrainment of evening and morning circadian oscillators. The authors argue for the incorporation of simulated natural photoperiods in studies of photoperiodism.

Temporal reorganization of the suprachiasmatic nuclei in hamsters with split circadian rhythms.

A dual oscillator basis for mammalian circadian rhythms is suggested by the splitting of activity rhythms into two components in constant light and by the photoperiodic control of pineal melatonin secretion and phase-resetting effects of light. Because splitting and photoperiodism depend on incompatible environmental conditions, however, these literatures have remained distinct. The refinement of a procedure for splitting hamster rhythms in a 24-h light-dark:light-dark cycle has enabled the authors to assess the ability of each of two circadian oscillators to initiate melatonin secretion and to respond to light pulses with behavioral phase shifting and induction of Fos-immunoreactivity in the suprachiasmatic nuclei (SCN). Hamsters exposed to a regimen of afternoon novel wheel running (NWR) split their circadian rhythms into two distinct components, dividing their activity between the latter half of the night and the afternoon dark period previously associated with NWR. Plasma melatonin concentrations were elevated during both activity bouts of split hamsters but were not elevated during the afternoon period in unsplit controls. Light pulses delivered during either the nighttime or afternoon activity bout caused that activity component to phase-delay on subsequent days and induced robust expression of Fos-immunoreactivity in the SCN. Light pulses during intervening periods of locomotor inactivity were ineffective. The authors propose that NWR splits the circadian pacemaker into two distinct oscillatory components separated by approximately 180 degrees, with each expressing a short subjective night.

Long-term reproductive effects of a single long day in the Siberian hamster (Phodopus sungorus).

Testicular regression was prevented or attenuated in Siberian hamsters exposed to a single 1- to 4-h extension of the 16-h photophase at 18 days of age and subsequently maintained in a short photoperiod (8L:16D) through Day 35. Testicular weights on Day 35 were not correlated with the duration of the active phase of wheel running or with the time of activity onset after transfer to the 8L:16D photoperiod. Wheel-running activity was not stably entrained to the light-dark cycle by 35 days of age. Progonadal effects of a single 33-h light pulse were greatest at 18 days of age, still evident at 30 days, but undetectable in older hamsters. In female hamsters, a single longer day at weaning was associated with increased fecundity several weeks later. Long photoperiods accelerated development of antral ovarian follicles, but exposure to males was necessary to induce ovulation before 60 days of age. The interval beginning shortly after weaning is one of heightened responsiveness to changes in day length (DL); exposure to increasing DL at this time may prolong the breeding season when DL decreases after the summer solstice. We suggest that the long-term effects of acute light treatments on reproduction are mediated by sustained changes in melatonin secretion induced by reprogramming of circadian oscillators.

Melatonin chimeras alter reproductive development and photorefractoriness in Siberian hamsters.

Nightly melatonin (MEL) durations > 8 h provoke gonadal regression and decreases in body mass, whereas signals < 7 h stimulate gonadal and somatic growth in male Siberian hamsters. The authors sought to determine the minimum frequency of short MEL signals sufficient to induce the long-day phenotype in several photoperiodic traits. D,L-propranolol (hereafter propranolol) injections shortened MEL signals on the night of treatment without altering MEL on the subsequent night; this permitted interpolation of short MEL signals at variable frequencies against a background of long MEL signals (chimeras). Hamsters kept in short days (10 h light/day, 10L) were injected with propranolol 6 h after dark onset for 28 consecutive weeks beginning at 30 days of age (Week 0) either every other day or once every 3, 6, or 9 days. Control animals were injected with saline or with propranolol during the light phase or were transferred to long days (16L) at Week 0. Hamsters in 16L underwent rapid gonadal development and increases in body mass and displayed summer pelage color, as did hamsters treated with propranolol every other day. Animals treated with propranolol less frequently than every other day uniformly maintained undeveloped gonads and winter-like body weights, but pelage color became proportionately darker with increased frequency of propranolol treatments. The onset of spontaneous testicular development in 10L was unaffected by propranolol injections. After termination of injections at Week 28, testicular regression was not observed in most 10L animals that previously had undergone spontaneous testicular development; however, 40% of hamsters that had been injected with propranolol every 3rd night did manifest the winter phenotype after Week 28. In an alternating sequence, short MEL signals completely override long signals and induce the summer phenotype. Threshold frequencies differ for MEL stimulation of long-day pelage and gonadal phenotypes. The timing and development of refractoriness to MEL does not depend in any simple manner on the number of long MEL signals or on the accumulation of a reaction product produced by long, and depleted by short, MEL signals.

Comparison of a lysed whole blood method to purified cell preparations for lymphocyte immunophenotyping: differences between healthy controls and HIV-positive specimens.

Although the majority of clinical laboratories now use a lysed whole blood (LWB) method for routine immunophenotyping, researchers wishing to perform other types of studies with lymphocytes from HIV+ patients may still need to use purified cell preparations, such as peripheral blood mononuclear cells (PBMC). A comparison study of the two methods was performed, using peripheral blood specimens from normal donors and from patients infected with the human immunodeficiency virus (HIV+). Reproducibility studies and several types of holding studies (both before and after specimen processing) were also performed. The results suggest that the two different methods of sample preparation have different effects upon abnormal patient specimens than those observed in healthy controls. Immunophenotyping results derived from the two different methods cannot be considered equivalent for the purposes of quantitating the presence of a particular type of cell.

Chronic progressive multiple sclerosis: changes in phenotype and function of T helper subsets.

We have measured pokeweed mitogen-induced IgG secretion by peripheral blood mononuclear cells obtained from two different groups of progressive multiple sclerosis (MS) patients. MS patients with chronic progressive active disease (CPMS-A) have higher IgG secretion than stable (burnt-out) patients (CPMS-S). We have also measured suppressor cell function and phenotyped the T helper cells of some CPMS-A patients. This group differed from CPMS-S and from controls: they had high IgG secretion, low suppression and their T helper phenotypes showed a high ratio of T helper/inducers over T suppressor/inducers.

Presence of multiple anti-phospholipid antibody specificities in a pediatric population.

Thrombotic related events are thought to be associated with the presence of anti-phospholipid antibodies (APA). However, the association of anti-cardiolipin antibody is much weaker than the association with antibodies to other phospholipids. Much of the literature equates antiphospholipid antibodies and anticardiolipin antibodies because of the relationship of APA and false positive tests for syphilis. However, recently the presence of antibodies to naturally occurring phospholipids other than cardiolipin have been reported. In fact, some investigators report that antibodies to phosphatidylserine appear to correlate more closely to disease processes than anti-cardiolipin antibodies. We describe here the presence of non-anti-cardiolipin antiphospholipid antibodies in a pediatric population that lack anti-cardiolipin antibodies and demonstrate the association of these antibodies with thrombotic disease. Antibodies to phosphatidic acid were the most prevalent and correlated (p < .001) with thrombotic disease and idiopathic thrombocytopenia purpura. The rank order of prevalence of antibodies to phospholipids was phosphatidic acid, phosphatidylglycerol, phosphatidylinosital, phosphatidylserine, cardiolipin and phosphatidylethanolamine. Antiphospholipid antibodies of the three major sera isotypes were present in the positive sera examined. These descriptive findings suggest that the significance of APA other than anti-cardiolipin antibodies in pediatric patients should be further investigated.

CD8+ T cell-mediated suppression of human immunodeficiency virus replication in older children with acquired immunodeficiency syndrome.

BACKGROUND: Suppression of HIV replication by CD8+ T cells and/or their products correlated with the survival of infants. We sought to elucidate the role of CD8+ T cell-mediated suppression in seven older children with AIDS. METHODS: After separation of each child's CD4+ and CD8+ T cells, three different HIV culture assays were performed: (1) patient CD4+ T cells and phytohemagglutinin (PHA)-stimulated donor peripheral blood mononuclear cells (PBMC); (2) patient CD8+ T cells added to the CD4+ T cells and the PHA-stimulated donor PBMC (to test for CD8-mediated T cell suppression of HIV); (3) patient CD8+ cells added across a semipermeable membrane to the CD4+ T cells and the PHA-stimulated donor PBMC [to determine whether the CD8 cells secreted a soluble factor(s) that suppressed HIV]. RESULTS: Cultures from four of seven children showed greater HIV replication with CD4 cells alone than with CD4 and CD8 cells together, demonstrating CD8 suppression; evidence of soluble suppression was also seen. Cultures from two of the seven children showed HIV replication and no evidence of CD8 cell suppression. Cultures from one of the seven children had no appreciable replication of HIV even after removal of CD8 cells. CONCLUSIONS: CD8-mediated suppression is present in at least some children with AIDS. Additional mechanisms may be operating to slow the progression of the disease.

Melatonin implants disrupt developmental synchrony regulated by flexible interval timers.

Siberian hamsters born into short daylengths near the end of the breeding season are reproductively inhibited from birth and delay gonadal maturation until the following spring. This vernal transition to a reproductive phenotype coincides with an abrupt increase in body weight, and both processes are triggered by an interval timing mechanism that becomes insensitive, or refractory, to short-day inhibition. It was previously demonstrated that hamsters born into simulated natural photoperiods in early August became photorefractory at later ages than hamsters born into September photoperiods. As a consequence of flexibility in the duration programmed by the interval timer, development of seasonal birth cohorts was synchronous with respect to the calendar date simulated by laboratory photoperiod. In the present study, hamsters were born into simulated August or September photoperiods. Hamsters from each cohort were given removable constant release melatonin implants to reversibly obscure the neuroendocrine representation of daylength between 3 and 9 weeks or 9-15 weeks of age. When control hamsters were given beeswax capsules throughout, August-born males were approximately 6 weeks older than September males at the onset of photorefractoriness as assessed by accelerated increases in body weight and testicular size. Females exhibited the same pattern in body weight. These measures were synchronized with respect to calendar date. Synchronization of cohorts was disrupted by melatonin capsules from 3-9 weeks of age but not by later implants. Melatonin implants altered synchronization by influencing the developmental trajectory of September-born hamsters without influencing the August cohort. These results demonstrate that the function of the interval timer underlying photorefractoriness is influenced by photoperiod and by melatonin. The endogenous pattern of melatonin signals adjusts the duration measured by the interval timer to insure that developmental milestones of seasonal cohorts are synchronized with environmental conditions.

Correction of neutropenia and hypogammaglobulinemia in X-linked hyper-IgM syndrome by allogeneic bone marrow transplantation.

X-linked hyper-IgM (X-HIM) syndrome is a primary immunodeficiency disease characterized by defects in both cellular and humoral immunity. X-HIM is caused by mutations in the gene for CD40 ligand (CD40L), a T cell membrane protein that mediates T cell-dependent immune functions. We report the case of a 6-year-old male with X-HIM due to an intronic mutation resulting in aberrant CD40L RNA splicing and absence of detectable CD40L protein. The patient had a history of multiple infectious complications and chronic neutropenia requiring treatment with recombinant granulocyte colony-stimulating factor, and underwent allogeneic bone marrow transplantation from an HLA-matched sibling donor. Following successful engraftment, T cell CD40L expression and immunoglobulin isotype switching were reconstituted and neutropenia resolved. Allogeneic bone marrow transplantation can correct neutropenia and reconstitute immune function in X-HIM.

Apoptosis occurs in isolated and banked primary mouse hepatocytes.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.

Three daily melatonin infusions alter gonadal development but not GnRH neuron number in the medial preoptic area or diagonal band of Broca in Siberian hamsters.

Among juvenile Siberian hamsters reproductive development is associated with an increased number of unipolar GnRH-immunoreactive neurons in the diagonal band of Broca and medial preoptic area. In the present study, GnRH neuron morphology was assessed in male juvenile hamsters which were treated with three daily melatonin (MEL) infusions to initiate or delay gonadal development. Hamsters gestated in short days were transferred to constant light (LL) at day 14 of age and infused on days 18-20 with MEL for 6 h/day. This treatment stimulated testis development compared to that in saline-infused controls. By contrast, testis growth was suppressed by three 12 h MEL infusions in long-day gestated hamsters in LL from day 14. The number of unipolar and bipolar GnRH neuron subtypes was the same irrespective of MEL infusion or preweaning photoperiod. Thus, gonadal response to three MEL infusions is independent of changes in GnRH neuron number.

Photoperiod and gonadal hormones influence odor preferences of the male meadow vole, Microtus pennsylvanicus.

Male meadow voles housed in a long photoperiod (14 h light/day, LP) preferred female to male odors, whereas males maintained in a short photoperiod (10 h light/day, SP) did not display preferences for odors of either sex. These odor-preference patterns matched those of free-living males during spring and autumn, respectively. The preference of LP male voles for female over male odors was eliminated by gonadectomy and reinstated by treatment with testosterone. In SP males, although gonadectomy did not affect odor choices, a preference for female odors was induced by testosterone treatment. Treatment with estradiol did not alter odor preferences of LP or SP males. In conjunction with previous result, the present findings suggest that hormonal responsiveness of neural substrates that control odor preferences are sexually dimorphic and may reflect sex differences in reproductive strategies.