Y chromosome--a tool in infertility studies of Latvian population.

UNLABELLED: Human Y chromosome is used as a tool in male infertility and population genetic studies. The aims of this research were to analyse the prevalence of Y chromosome microdeletions among infertile Latvian men, and to identify possible lineages of Y chromosome that may be at increased risk of developing infertility. A study encompassed 105 infertile men with different spermatogenic disturbances. Deletions on Y chromosome were detected in 5 out of 105 (approximately 5%) cases analysed in this study. Three of them carried deletion in AZFc region and two individuals had AZFa + b + c deletion. Study of Y chromosome haplogroups showed that N3a1 and R1a1 lineages were found less frequently in the infertile male group compared to ethnic Latvian group, however K* cluster was predominantly found in infertile male Y chromosomes. CONCLUSIONS: 1) Our study advocates running Y chromosome microdeletion analyses only in cases of severe form of infertility; 2) Y chromosome haplogroup analysis showed statistically significant tendencies that some haplogroups are more common in ethnic male group, but others are more common in infertile males.

Mitochondrial DNA portrait of Latvians: towards the understanding of the genetic structure of Baltic-speaking populations.

Mitochondrial DNA (mtDNA) variation was investigated in a sample of 299 Latvians, a Baltic-speaking population from Eastern Europe. Sequencing of the first hypervariable segment (HVS-I) in combination with analysis of informative coding region markers revealed that the vast majority of observed mtDNAs belong to haplogroups (hgs) common to most European populations. Analysis of the spatial distribution of mtDNA haplotypes found in Latvians, as well as in Baltic-speaking populations in general, revealed that they share haplotypes with all neighbouring populations irrespective of their linguistic affiliation. Hence, the results of our mtDNA analysis show that the previously described sharp difference between the Y-chromosomal hg N3 distribution in the paternally inherited gene pool of Baltic-speaking populations and of other European Indo-European speakers does not have a corresponding maternal counterpart.

Morphology and ultrastructure of rat hippocampal formation after i.c.v. administration of N-acetyl-L-aspartyl-L-glutamate.

N-Acetyl-L-aspartyl-L-glutamate (NAAG) is one of the most abundant neuroactive compounds in the mammalian CNS. Our recent observations have suggested that NAAG administered into rat cerebral ventricles can cause neuronal death by apparently excitotoxic mechanisms that can be antagonized by the N-methyl-D-aspartate-receptor blockers and by ligands of metabotropic glutamate receptor of Group II. Therefore, the principal aim of the present study has been to use quantitative morphology, electron microscopy and terminal deoxynucleotidyl transferase-mediated biotin dUTP nick-end labeling to study a dose- and time-dependence as well as regional distribution of neurodegeneration in hippocampi of rats after the intraventricular infusion of 0.25 micromol NAAG/ventricle and of equimolar doses of L-glutamate (L-GLU) and N-acetyl-L-aspartate (NAA), breakdown products of NAAG. The degenerative changes were observed after the infusion of 0.25 and 1.25 micromol of NAAG/ventricle, but not when a dose of 0.05 micromol of NAAG/ventricle was injected into each lateral cerebral ventricle. With a dose of 0.25 micromol of NAAG/ventricle the number of degenerated neurons reached a maximum on the fourth day after the infusion. The neuronal damage following bilateral administration of 0.25 micromol of NAAG/lateral cerebral ventricle exhibited features of a delayed neuronal degeneration, expressed mainly in the layer of dentate granule neurons. The degeneration was characterized on the basis of ultrastructural appearance and DNA-fragmentation. The morphological changes caused by L-glutamate and NAA were much smaller than those observed after the administration of NAAG and displayed a different pattern of regional distribution. The present findings suggest that NAAG can cause a loss of hippocampal neurons in vivo, apparently resulting from the neurotoxicity of NAAG itself.

Regional distribution and pharmacological characteristics of [3H]N-acetyl-aspartyl-glutamate (NAAG) binding sites in rat brain.

Autoradiographical studies revealed that 10 nM [3H]N-acetyl-aspartyl-glutamate (NAAG) labelled grey matter structures, particularly in the hippocamus, cerebral neocortex, striatum, septal nuclei and the cerebellar cortex. The binding was inhibited by (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG IV), an agonist at group II metabotropic glutamate receptors (mGluR II). (RS)-alpha-Methyl-4-tetrazolylphenylglycine (MTPG), (RS)-alpha-cyclopropyl-4-phosphonoglycine (CPPG) and (RS)-alpha-methylserine-O-phosphate monophenyl ester (MSOPPE), all antagonists at mGluR II and mGluR III, also inhibited [3H]NAAG binding. Other inhibitors were (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a broad-spectrum mGluR agonist with preference for groups I and II and the mGluR I agonists/mGluR II antagonists (S)-3-carboxy-4-hydroxyphenylglycine (3,4-CHPG) and (S)-4-carboxy-3-hydroxyphenylglycine (4,3-CHPG). Neither the mGluR I specific agonist (S)-dihydroxyphenylglycine nor any of the ionotropic glutamate receptor ligands such as kainate, AMPA and MK-801 had strong effects (except for the competitive NMDA antagonist CGS 19755, which produced 20-40% inhibition at 100 microM) suggesting that, at low nM concentrations, [3H]NAAG binds predominantly to metabotropic glutamate receptors, particularly those of the mGluR II type. Several studies have indicated that NAAG can interact with mGluR II and the present study supports this notion by demonstrating that sites capable of binding NAAG at low concentrations and displaying pharmacological characteristics of mGluR II exist in the central nervous tissue. Furthermore, the results show that autoradiography of [3H]NAAG binding can be used to quantify the distribution of such sites in distinct brain regions and study their pharmacology at the same time.

Neurotoxicity of NAAG in vivo is sensitive to NMDA antagonists and mGluR II ligands.

N-Acetyl-aspartyl-glutamate (NAAG), an agonist at Group II metabotropic glutamate receptors (mGluR II), also activates the NMDA-type of ionotropic glutamate receptors and, at high micromolar concentrations, has previously been shown to induce neuronal cell death. In the present study we have morphologically quantified the neurotoxic action of intracerebroventricularly administered NAAG on the hippocampal formation and compared it to the action of the selective endogenous NMDA agonist quinolinic acid. Finally, we examined whether the action of NAAG can be modified by NMDA receptor antagonists and mGluR II ligands. NAAG-induced neurodegeneration was found to be less severe than that induced by quinolinate. It was prevented by inhibitors of NMDA receptors and also by an mGluR II agonist (DCG IV) but not by an mGluR II antagonist (EGlu).

Quinolinic acid enhances permeability of rat brain microvessels to plasma albumin.

Several studies have established that increased cerebrospinal fluid (CSF) levels of quinolinic acid (QUIN), a macrophage/microglia-derived excitotoxin with N-methyl-D-aspartate (NMDA)-receptor affinity, may reflect abnormal blood-brain barrier (BBB) function in patients with acquired immunodeficiency syndrome (AIDS) dementia complex, exhibiting a relationship to their clinical and neurological status. This study was aimed to evaluate whether QUIN (250 nmol/0.25 microl/ventricle) infused into both lateral cerebral ventricles permeates adult rat brain microvessels to plasma albumin. Possible BBB dysfunction was examined 4 days after the intracerebroventricular (i.c.v.) infusion of QUIN by measuring plasma albumin extravasation using rocket immunoelectrophoresis. The i.c.v. infusion of QUIN failed to increase the extracellular tissue concentration of albumin in the entorhinal cortex, but significantly higher levels were found in the hippocampus proper (but not in the subiculum region and dentate gyrus) and in the striatum. To evaluate the possible relationship between plasma protein extravasation and QUIN-induced tissue necrosis, we quantified neuronal death in the rat hippocampal formation (subiculum, CA1/CA3 areas of the hippocampus proper, dentate gyrus). We found significantly higher tissue levels of plasma albumin in the hippocampus proper, in which the CA1 area exhibited the highest neuronal loss while the low rate of neuronal death was not accompanied by significant albumin extravasation in the dentate gyrus. However, in case of the subiculum, in which the neuronal loss reached comparable values to those in the CA1 area, we did not find significant enhancement of plasma albumin leakage into this area. The regional differences in brain microvascular permeability may depend on the density of NMDA receptors in the multicellular capillary barrier, but the differences in neuronal death may also reflect an involvement of NMDA receptors in neuronal membranes. We conclude that increased CSF QUIN levels evoke a dysfunction of the BBB that may only partially be related to sites with pronounced neuronal damage in the rat brain regions susceptible to NMDA-receptor mediated toxicity.

Relationship among mediators, inflammation, and volume history with antigen versus hyperpnea challenge in guinea pigs.

Paralyzed mechanically ventilated guinea pigs constricted to a similar degree by either isocapnic hyperpnea or antigen challenge display significantly different lung resistance (RL) volume history responses to a deep breath. We compared bronchoalveolar lavage (BAL) mediator profiles, BAL total protein concentrations, and tissue histopathology of antigen-constricted (AC), hyperpnea-constricted (HC), and control guinea pigs to determine whether patterns of volume history near peak constriction could be related to specific patterns of lung mediators, indices of microvascular leakage, or severity of tissue inflammation assessed pathologically. Methacholine constricted (MC) animals served as a second control group for assessing the effects of direct smooth-muscle contraction on indices of inflammation and volume history responses. Our results show that despite similar baseline and postchallenge RL, HC and MC animals displayed significant constriction reversal after a deep lung inflation, whereas AC animals did not. BAL concentrations of prostaglandin D2(PGD2), thromboxane B2 (TxB2), and leukotriene C4/D4/E4 (slow reacting substance of anaphylaxis, SRSA) were significantly elevated in both AC and HC animals compared with control and MC animals, with AC and HC BAL differing only with respect to PGD2 values (AC 2.4-fold higher). BAL total protein in AC animals was significantly greater than in HC, MC, and control animals. Histopathology showed significant peribronchial and interstitial cellular inflammation in AC animal specimens, whereas specimens from HC animals had little or no inflammation. Differences in volume history responses observed between equally constricted AC, HC, and MC animals may be due to differences in airway and/or parenchymal microvascular leak and cellular inflammation.

Effects of capsaicin on mechanical, cellular, and mediator responses to antigen in sensitized guinea pigs.

To assess the role of tachykinins (TKs) in immediate hypersensitivity allergic reactions in guinea pigs (GPs), we compared airway mechanics and bronchoalveolar lavage (BAL) cell and inflammatory mediator profiles in three groups of GPs after ovalbumin aerosol challenge: (1) saline-sensitized, noncapsaicinized (control) (n = 9); (2) ovalbumin-sensitized, noncapsaicinized (OS) (n = 9); (3) ovalbumin-sensitized capsaicinized (OSC) (n = 9). Lung resistance (RL), dynamic elastance (EL), BAL cell counts, histamine, and eicosanoid mediator levels were measured at baseline on Day 1, and then on Day 14 after aerosolized antigen challenge. We found significant increases on Day 14 compared with Day 1 in the following: (1) postchallenge RL and EL in OS and OSC GPs, but not in control GPs; (2) BAL total cells and red cells in OS and OSC GPs; (3) BAL prostaglandin D2 (PGD2) thromboxane B2 (TxB2), sulfidopeptide leukotrienes (LTC4/D4/E4), and histamine in OS and OSC animals. Further, when data from all GPs were considered in distributed fashion, we noted positive linear correlations between peak postchallenge RL versus BAL concentrations of each of the following: PGD2, PGF2 alpha, TxB2, LTC4/D4/E4, leukotriene B4 (LTB4), and histamine. We found no significant differences in mediator or cellular responses between OS and OSC GPs. To verify that our method of capsaicinization resulted in TK depletion from the lungs of OSC GPs, substance P (SP) and neurokinin A (NKA) lung tissue levels were measured by ELISA in seven other animals, four treated with capsaicin using the same protocol and three treated with diluent.(ABSTRACT TRUNCATED AT 250 WORDS)

Baseline lung function and inflammatory indices in guinea pigs: effects of growth, repeated lavage, and orotracheal intubation.

We examined the relationships between baseline lung function, weight, and indices of inflammation in bronchoalveolar lavage (BAL) liquid in unsensitized, anesthetized guinea pigs (GPs) and evaluated a model for performing orotracheal intubation and BAL without having to sacrifice the animal. Thirty-six GPs were anesthetized and orotracheally intubated. Lung resistance (RL) and elastance (EL) were measured. BAL was then performed with sterile saline, and the animal permitted to cover. White blood cells and differentials, total protein (TP), histamine, prostaglandins (PGs) D2, F2a, E2, leukotrienes (LTs) B4, and C4/D4/E4 were measured in the BAL liquid as indices of inflammation. Body weight (and by inference, age) directly correlated with BAL concentrations at TP and PGD2 and inversely correlated with LTC4/D4/E4 and % eosinophils. Significant correlations were found between PGD2 vs. TP (r = 0.54, p less than .001), histamine vs. % eosinophils (r = 0.45, p = 0.03), and % macrophages vs. % neutrophils (r = -0.76, p less than .001). RL and EL did not correlate with BAL measurements of cells or mediators. A second group of 9 animals was lavaged, permitted to recover, and relavaged 2 weeks later, with no significant changes noted in lung mechanics, BAL cell profiles, or mediators between study days. These data demonstrate that some baseline BAL inflammatory indices in the GP correlate well with body weight and with each other and that baseline BAL measurements of inflammatory mediators may vary with size or age of the animal, an important consideration in studies using the GP for BAL measurements of inflammatory mediators. The data further document the reproducibility and feasibility of reintubating and lavaging the same animal over a period separated by 2 weeks without having to sacrifice the animal.

Assessment of bronchoalveolar cell and mediator response to isocapnic hyperpnea in asthma.

The purpose of this study was to test the hypothesis that mediators and cells associated with bronchoconstriction or inflammation are locally synthesized and/or released in the airways of asthmatic subjects in response to isocapnic hyperpnea (ISH). Seven atopic, mildly asthmatic subjects were studied. Baseline measurements were reported previously and included forced expiratory volumes, flow rates, bronchoalveolar lavage (BAL), and methacholine reactivity. Approximately 1 yr later, spirometry and BAL were repeated, but BAL was performed immediately after ISH challenge. As indices of inflammation, BAL measurements were made of eosinophils, neutrophils, epithelial cells, leukotrienes B4, C4, D4, and E4, prostaglandins D2, E2, and F2 alpha, thromboxane B2, histamine, and total protein. Compared with baseline, ISH was associated with higher BAL concentrations of the following: leukotriene B4 (10 versus 121 pg/ml, p = 0.02), leukotrienes C4/D4/E4 (46 versus 251 pg/ml, p = 0.02), eosinophils (0.8 versus 2.2%, p = 0.04), and epithelial cells (2.1 versus 6.1%, p = 0.05). Trends toward significant increases were seen in BAL concentrations of neutrophils and prostaglandin D2. No statistically significant increases were found in BAL measurements of total protein, histamine, prostaglandins E2 or F2 alpha, thromboxane B2, lymphocytes, or macrophages. The magnitude of the response to ISH, as measured by change in FEV1, did not correlate with BAL levels of cells or mediators. This study indicates that ISH, even in mildly asthmatic subjects, is associated with airway increases in a spectrum of bronchoactive mediators and inflammatory cells, supporting the observations of others that antagonists of a single mediator are unlikely to have major clinical effectiveness in ISH or exercise-induced asthma.

Bronchoalveolar lavage cell and mediator responses to hyperpnea-induced bronchoconstriction in the guinea pig.

We studied the association between bronchoconstriction and bronchoalveolar lavage (BAL) cell and mediator profiles in unsensitized guinea pigs (GP) after hyperpnea to determine whether eicosanoids or histamine are released during hyperpnea-induced bronchoconstriction (HIB). Twelve animals were challenged with warm, moist (WM) air (T = 35 degrees C, relative humidity = 91 to 94%), 14 with room dry (RD) air (T = 25 degrees C, relative humidity less than 2.1%), and 18 with cold, dry (CD) air (T = 7 degrees C, relative humidity less than 2.1%). Lung resistance (RL) and elastance (EL) were recorded at baseline and at 2-min intervals after hyperventilation. Challenges were terminated either when a greater than or equal to 100% increase in RL was observed postchallenge or after completion of a 135 breaths/min challenge if RL did not increase. BAL was performed, and samples were analyzed for total cells, white cell and epithelial cell differentials, total protein concentration, and mediator content.(ABSTRACT TRUNCATED AT 250 WORDS)

Responsiveness, inflammation, and effects of deep breaths on obstruction in mild asthma.

Using cellular and biochemical characteristics of bronchoalveolar lavage (BAL) liquid as an index of inflammation, we examined the relationships between change of airway caliber after a deep inhalation (DI), degree of base-line airway hyperresponsiveness, and peripheral airway inflammation in a group of 16 atopic asymptomatic mild asthmatics and 6 normal subjects. Compared with normal subjects, asthmatics demonstrated 1) significantly higher BAL concentrations of histamine, total protein, the sulfidopeptide leukotrienes (SRS-A), and leukotiene B4; 2) a decrease in specific airway conductance (sGaw) with a DI at base line vs. an increase in normal subjects (before vs. after percent change in sGaw, -10 vs. 12, P less than 0.05); and 3) no significant difference in BAL total cell count or leukocyte differential. Significant correlations were demonstrated between 1) percent of BAL eosinophils vs. degree of airway hyperresponsiveness; 2) base-line level of airway obstruction vs. degree of hyperresponsiveness; 3) effects of a DI vs. BAL concentrations of eosinophils, total protein, and histamine; 4) base-line forced expired volume in 1 s vs. BAL concentrations of total protein and histamine; and 5) BAL concentrations of the various mediators with each other. These data support the notion that 1) the response to a DI in mild, stable asthmatics represents a physiological indicator of peripheral obstruction because of inflammation and 2) this inflammation is associated with increases in several known mediators of airway inflammation and hyperreactivity.

Aerosol vs injected epinephrine in acute asthma.

Twenty-five patients with acute asthma were randomized prospectively into one of two double-blind treatment regimens: regimen 1 consisted of subcutaneous epinephrine combined with aerosol placebo; regimen 2 consisted of aerosol epinephrine with injected placebo. In patients with severe airway obstruction (peak expiratory flow rate less than 120, or less than 25% of predicted normal), parenteral epinephrine was superior to aerosol (P less than 0.005) at the end of one hour. However, in patients with mild to moderate asthma (PEFR greater than 120), injected and inhaled epinephrine were of equal efficacy, with the aerosol producing fewer side effects (P less than 0.001).

Differentiation of human leukemic from normal lymphocytes by Limulus serum agglutination.

Human peripheral chronic lymphocytic leukemic lymphocytes and a line (B411-4) of cultured human B cells are agglutinated by Limulus serum to a significantly higher titer and score than peripheral normal human lymphocytes or a line (MOLT-4-F) of cultured human T cells.