HLA-DQA2 (DX alpha) polymorphism and insulin dependent diabetes

A certain HLA-DQA2 locus TaqI fragment, DX“U”, has been reported to be associated with insulin-dependent diabetes mellitus (IDDM). Reports of various studies in this vein have ranged from stating that the association of DQA2“U” with IDDM exists even among subjects positive for HLA-DR3 and -DR4 to stating that the association of DQA2“U” with diabetes can be attributed to linkage disequilibrium between with DQA2“U” and some component(s) on the affected haplotypes. Using a synthetic 97-base probe corresponding to a portion of an intron of DQA2, in a Southern blot analysis of IDDM and control subjects from Wisconsin, we were able to confirm the association of DQA2“U” with diabetes. However, among DR3 subjects there was no significant association between DQA2“U” and diabetes (p = 0.26). Although there was a (nonsignificant) association of IDDM with DQA2“U” among DR4-positive subjects (p = 0.14), this can be completely attributed to linkage disequilibrium between DQA2“U” DQw8. We also sequenced most of the second exon (corresponding to the 1 domain of the DQA2 glycoprotein) from five individuals that were homozygous for either DQA2“U” or DQA2“L.” The only polymorphisms observed were a “silent” mutation at position 36 and one example of a difference that would result in a change of amino acid at position 41.     

Correlation of estrogen receptor beta gene polymorphisms with spinal bone mineral density in peri- and post-menopausal Greek women

Estrogens play a significant role in bone physiology. Their action is mainly exerted through their receptors. Estrogen receptor alpha (ER) plays a major role in bone homeostasis and there is evidence suggesting that estrogen receptor beta (ERβ) has also an effect on BMD.

We investigated the possible effect of two ERβ gene polymorphisms on spinal bone mineral density (BMD) and metabolic bone markers in Greek women.

Spine BMD as well as biochemical bone markers were measured in 147 healthy peri- and post-menopausal women [mean age (S.D.) 54 (7.9) years]. Genotyping was performed for two restriction fragment length polymorphisms (RFLPs) of ERβ gene, RsaI in exon 5 and AluI in exon 8. For each polymorphism studied the cohort was divided into two groups: the “wild-type” group (RR and AA, respectively) and the “carrier” group including subjects with at least one allele with the restriction site (Rr&rr and Aa&aa, respectively).

The distribution of RsaI genotypes was RR: 91.2% (n = 134), Rr: 8.2% (n = 12), and rr: 0.6% (n = 1) and of AluI genotypes AA: 36.7% (n = 54), Aa: 57.2% (n = 84), and aa: 6.1% (n = 9). No linkage disequilibrium was found between the two polymorphic sites studied. Spine BMD did not differ significantly in the two groups of either polymorphism, after adjusting for age, weight, height, and years since menopause [mean BMD (S.D.) for RR 0.841 (0.17) g/cm² versus Rr&rr 0.798 (0.13) g/cm², p = 0.25, and mean BMD (S.D.) for AA 0.828 (0.16) g/cm² versus Aa&aa 0.848 (0.17) g/cm², p = 0.32]. No significant differences were noted in metabolic bone markers except for a marginal difference of RR versus Rr/rr in urinary hydroxyproline/creatinine ratio [median (IQR) 3.88 (6.04) μmol/mmol in RR versus 8.2 (4.32) μmol/mmol in Rr/rr, p = 0.05]. Furthermore, no interaction between the two polymorphisms on BMD was found.

In conclusion, in a Greek female post-menopausal population, the two ERβ gene polymorphisms were not associated with BMD, or metabolic bone markers.     

Restorative potential of dopaminergic grafts in presence of antioxidants in rat model of Parkinson's disease

Free radical mediated damage has been reported to contribute significantly towards low survival (5–10%) of grafted dopaminergic neurons, post transplantation. In the present study, an attempt has been made to explore the neuroprotective potential of the combination of two major antioxidants ascorbic acid (AA) and glutathione (GSH) on ventral mesencephalic cells (VMC) and nigral dopamine (DA) neurons when co-transplanted together with VMC in rat model of Parkinson's disease (PD). GSH and AA have been reported to act co-operatively in the conditions of oxidative stress thereby helping in maintaining the cellular GSH/GSSG redox status. Functional recovery was assessed 12 weeks post transplantation, where a significant restoration (p < 0.001) in D-amphetamine induced circling behavior (62%), spontaneous locomotor activity (SLA; 64%), dopamine–D₂ receptor binding (63%), dopamine (65%) and 3,4-dihydroxy phenyl acetic acid (DOPAC) level (64%) was observed in co-transplanted animals as compared to lesioned and VMC alone grafted rats. VMC and GSH + AA co-transplanted animals exhibited a significantly higher surviving TH-immunoreactive (TH-ir) neurons number (p < 0.01), TH-ir fibers outgrowth (p < 0.05) in striatal graft and TH-ir neurons in substantia nigra pars compacta (SNpc) (p < 0.01), as compared to VMC alone transplanted rats. An attempt was made to further confirm our in vivo observations through in vitro experiments where following in vitro exposure to 6-OHDA, a higher cell survival (p < 0.01), TH-ir cell counts (p < 0.001) and DA and DOPAC levels (p < 0.01) were also observed in 8-day-old VMC culture in presence of GSH + AA as compared to VMC cultured in absence of antioxidants. The results suggest that GSH + AA when co-transplanted with VMC provide higher restoration probably by increasing the survival of grafted VMC and simultaneously supporting nigral TH-immunopositive neurons in rat model of PD.     

The association between mannose-binding lectin gene polymorphism and rheumatic heart disease

Mannan-binding lectin (MBL) is an innate pattern recognition molecule known to play a key role in pathogen clearance. As MBL2 gene polymorphism is associated to an increased susceptibility to infection, we aimed to determine genetic variations in the MBL2 gene in rheumatic heart disease (RHD). Genetic variations in the promoter and exon 1 region of the MBL2 gene were analyzed in 107 patients with RHD and 105 controls by real-time polymerase chain reaction. The frequency of MBL2* A/A genotype was significantly higher in RHD patients (71/107, 66.36% vs 52/105, 49.52%, p ≤ 0.02, OR = 1.99, 95% CI, 1.15–3.50). A/A genotypes were associated with higher levels of MBL in RHD compared with controls with the same genotype (p ≤ 0.004). The frequency of HYPA/HYPA, HYPA/LYQA, and LYQA/LYQA haplotypes was also increased in RHD (p ≤ 0.03, OR = 1.98, 95% CI, 1.05–3.73). However, the frequency of MBL2 variant alleles (termed “O”) was lower among patients (39/214, 18.2% vs 63/210, 30.0%, p ≤ 0.006, OR = 0.52, 95% CI, 0.33–0.82), which was also seen for O/O genotypes (3/107, 2.8% vs 10/105, 9.5%, p ≤ 0.05, OR = 0.27, 95% CI, 0.07–1.03). This data suggests a role for MBL genotypes in the susceptibility to RHD. However, it still remains unclear whether A/A homozygosity is a risk factor for RHD or rheumatic fever itself.     

Analysis of post-transplant immune status in recipients of liver/bone marrow allografts

The aims of this study were to assess the effect of donor bone marrow infusion on the reactivity of recipient peripheral blood lymphocytes (PBL) to mitogen and to donor and third-party cells after primary liver allotransplantation and to identify any correlation between altered immunoreactivity and HLA mismatches, occurrence of rejection, and immunosuppression. The immunoreactivity of recipient PBL toward frozen donor splenocytes was evaluated in mixed lymphocyte culture (MLC) (n = 29) and cell-mediated lympholysis (CML) (n = 27) assays in time intervals ranging from 0.7 to 27 months after transplant. Overall, the mean anti-donor MLC stimulation index (SI) fell from 25.6 ± 5.2 preoperatively to 4.8 ± 1.7 after transplantation (p < 0.002), with 14 out of 29 (48.3%) patients developing donor-specific MLC hyporeactivity. HLA class II mismatches were significantly associated with recipient post-transplant immune profile (p < 0.05): MLC donor specific hyporesponsiveness was observed in 70%, versus 37% of patients who shared a class II antigen, versus those that did not. Of the control group, 61.1% developed donor-specific nonreactivity versus 27.2% in the donor bone marrow cells (DBMC) group (p = 0.02). Donor-specific CML hyporeactivity was observed after transplantation, independent of DBMC infusion, with mean percentage values of pre- and post-transplant donor-specific lysis of 22.4% ± 4.1% versus 3.1% ± 1.6%, p = 0.0004, respectively. Our results suggest that DBMC infusion favors development of nonspecific MLC hyporesponsiveness to donor and third-party alloantigen, with maintenance of reactivity to mitogen and no additional effect on T-cell cytotoxicity.     

Alleles of the IL12B 3'UTR associate with late onset of type 1 diabetes

Carriage of a polymorphism in the 3′untranslated region of the IL12B gene encoding IL-12p40 was investigated in subjects with type 1 diabetes mellitus stratified by age at diagnosis (n = 648) and compared with a population-based control cohort (n = 246) residing in Western Australia. DNA samples were genotyped by polymerase chain reaction-restriction fragment length polymorphism or pyrosequencing. The C allele was more common in patients diagnosed after age 16 years than in controls (29% vs 17%, OR = 2.0, 95% CI = 1.4–2.7, p = 0.00003) or than in patients diagnosed when younger age 16 years (29% vs 22%, OR = 1.4, 95% CI = 1.1–1.9, p = 0.01). This reflected increases in homozygous and heterozygous carriage of the C allele. Heterozygosity was associated with a delayed disease in the late-onset diabetics (p = 0.005; Student’s t-test). The effects of IL12B 3′untranslated region alleles on type 1 diabetes mellitus may reflect different levels of p40 available to form p40 homodimer, IL-12 (p35p40), and IL-23 (p19p40).     

Delayed Acquisition of Behavioral and Hippocampal Responses During Jaw Movement Conditioning in Aging Rabbits

Young (3–7 months; n = 7) and aging (40–49 months; n = 7) rabbits (Oryctolagus cuniculus) were classically conditioned in a trace jaw movement paradigm (300 ms tone, 450 ms trace, 200 ms intraoral water) after implantation of electrodes into area CA1 of dorsal hippocampus. Aging rabbits took significantly more trials to reach a behavioral criterion of 8 conditioned responses in any 9 consecutive trials than young rabbits (p = 0.04), and their conditioned, but not unconditioned, jaw movement responses were of a lower frequency than those of young rabbits (p = 0.02). Early in training, aging rabbits’ hippocampal responses were significantly smaller just before water onset than corresponding responses of young rabbits (p = .03). The magnitude of this response was negatively correlated with trials to criterion (r = −0.60, p = 0.03). These results are interpreted in terms of age-related differences in the hippocampal contribution to jaw movement learning.     

Soluble HLA Class I Antigens in Patients with Type I Diabetes and Their Family Members

Our objective was to study a possible contribution of MHC genes to S-HLA-I secretion in patients with Type I diabetes. Quantitatively, we used a highly sensitive enzyme-linked immunoassay to measure S-HLA-I in the serum of a total of 39 patients with Type I diabetes, as well as 36 kinships of 12 diabetic patients and 82 normal individuals with known HLA-phenotypes.

S-HLA-I levels were abnormally elevated in patients or their non-diabetic relatives compared to normal controls (p < 0.0009). No complete HLA-haplotype had been identified to be correlated with high or low S-HLA-I secretion. Only the HLA-A23 or A24 (splits of HLA-A9) positive individuals sera were found to contain high S-HLA-I concentrations in all populations studied. The difference in S-HLA-I levels of HLA-A24 patients (n = 4) or their HLA-A24 positive non-diabetic relatives (n = 10) to the group of HLA-A24 normal controls (n = 15) was statistically highly significant (p < 0.0005 and p < 0.0009, respectively). The results suggests that HLA-A24 may confer additional independent risk for the disease expression in male children but not in female siblings. Nevertheless, the data implies that the patients or their non-diabetic relatives carrying the HLA-A24 have increased risk of developing ICA associated with high S-HLA-I levels compared to HLA-A24 negative probands or their kinships with low levels of S-HLA-I. This effect occurred irrespective to other diabetes related HLA-DR alleles.

In summary, the results show a pronounced genetic heterogeneity of Type I diabetes with MHC control of the expression of S-HLA-I and possible involvement of hormonal factors that might potentiate a specific synthesis of S-HLA-I. The findings have implications for identifying individuals with a possible risk for developing the disease.     

Potential influence of interleukin-1 haplotype IL-1 beta-511*T-IL-1RN*1 in conferring low risk to middle third location of esophageal cancer: a case-control study

Interleukin (IL)-1 gene polymorphisms affect several inflammatory diseases, including cancer. Therefore, we studied genetic association of biallelic (-511C>T) polymorphism of IL-1β and 86-bp VNTR polymorphism of IL-1RN in 159 patients with esophageal cancer (EC) and 194 age- and gender-matched healthy controls. Genetic analysis for IL-1 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism. The frequencies of IL-1β (-511C>T) and IL-1RN (variable number tandem repeat) genotypes, alleles, and haplotypes did not differ significantly between patients and controls. However, IL-1β -511TT genotype and T1+ haplotype combination illustrated low risk for disease at the middle third location of the tumor (odds ratio [OR] = 0.27; 95% confidence interval [CI] = 0.11–0.62; p = 0.002; OR = 0.462; 95% CI = 0.253–0.845, p = 0.01). In conclusion, subjects with IL-1β -511TT genotype or IL-1β*T-IL-1RN*1 (T1) haplotype had lower risk for middle third tumor location of EC in a northern Indian population.     

Neural dynamics in cortex-striatum co-cultures-II. Spatiotemporal characteristics of neuronal activity

Neural dynamics in organotypic cortex-striatum co-cultures grown for three to six weeks under conditions of dopamine deficiency are described. Single neuron activities were recorded intra- and extracellularly, and spatiotemporal spreading of population activity was mapped using voltage-sensitive dyes. The temporal properties of spike firing were characterized by interspike interval histograms, autocorrelation and crosscorrelation.

Cortical pyramidal neurons (n = 40) showed irregular firing with a weak tendency to burst or to oscillate. Crosscorrelations revealed strong near-coincident firing and synaptic interactions. Disinhibition was a notable feature in a strongly firing cortical interneuron. Cortical activity spread in the co-culture, thus inducing an overall, homogeneous depolarization in the striatal part. Striatal cells were divided into principal cells and type I and II secondary cells. Principal cells (n = 40) were similar to those reported previously in vivo. Spiking activity ranged from irregular spiking at very low rates to episodic bursting, with an average burst duration of 1 s. Interspike intervals were single-peaked. Intracellular recordings revealed characteristic, long-lasting subthreshold depolarizations (“enabled state”) that were shortened by local muscarinic receptor blockade. During prolonged time periods in the “enabled state”, locally applied bicuculline induced strong firing in most principal neurons. Striatal secondary type I neurons (n = 25) showed high spiking rates, single- and double-peaked interval histograms and low-threshold, short-lasting stereotyped bursting activity and occasional rhythmic bursting. The firing of these neurons was increased by bicuculline. Crosscorrelations showed synchronization of these cells with principal cell activity. Secondary type II neurons (n = 15) revealed tonic, irregular firing patterns similar to cortical neurons, except with occasional firing in doublet spikes.

We conclude that under conditions of dopamine deficiency in corticostriatal co-cultures (i) the cortex induces the “enabled” state and typical bursting mode in striatal principal neurons; (ii) principal neurons are strongly inhibited during the “enabled” state; (iii) muscarinic activity, presumably from tonically active striatal cholinergic interneurons, stabilizes the “enabled” state; (iv) striatal GABAergic interneurons receive synaptic inhibition and take part in synchronized activity among striatal principal cells. Our results favor the view of the striatum as a lateral inhibition network.     

Paroxysmal nocturnal hemoglobinuria: significant association with specific HLA-A, -B, -C, and -DR alleles in an Italian population

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p ≤ 0.05) and DRB1*01 (p ≤ 0.05) with the linked DQB1*0501 (p ≤ 0.01) alleles, has been observed. Notably, a fourfold increase of the haplotype B*1402, Cw*0802 (p < 0.0005) and a 15-fold increase of the Mediterranean haplotype A*33, B*1402, Cw*0802, DRB1*0102, DQB1*0501 (p < 0.005) was also revealed. This association may provide new insights into the autoimmune pathogenesis of PNH.     

Molecular identification of 13 new enterovirus types, EV79-88, EV97, and EV100-101, members of the species Human Enterovirus B

Paired PBMCs and plasma samples from 34 HIV-infected patients were studied to verify the relationship between coreceptor use based on genotyping of V3 region of HIV-1 envelope gp120 and biological phenotype with virus isolation and subsequent correlation to clinical characteristics. The “11/25” rule, geno2pheno and PSSM were compared. All SI patients were HIV-1 subtype B (p = 0.04) and had a lower CD4 count than NSI patients (p = 0.01), while no differences were observed in mean HIV-RNA _log (p = 0.6). SI phenotype was not associated with AIDS-defining events (p = 0.1) or with concurrent antiretroviral therapy (p = 0.4). With geno2pheno, which shows the highest sensibility (83%), an X4 or X4/R5 genotype in PBMC DNA was also associated to B-subtype and lower CD4 count (p = 0.01) compared to R5 isolates. Based on plasma RNA sequences, the predicted coreceptor usage agreed with PBMC DNA in 79% of cases with the “11/25” rule, 82% with geno2pheno, and 82% with PSSM. A X4 virus in plasma (but not in PBMCs) was significantly associated with HAART in all three methods (p = 0.01 for “11/25” rule, p = 0.01 for geno2pheno and p = 0.03 for PSSM). Due to viral mixtures and/or difficulties in genotype interpretation, current V3 sequence-based methods cannot accurately predict HIV-1 coreceptor use.     

Endogenous retroviral long terminal repeats of the HLA-DQ region are associated with susceptibility to insulin-dependent diabetes mellitus

HLA-DQ genes are the main inherited factors predisposing to IDDM. This gene region harbors long terminal repeat (DQ LTR) elements of the human endogenous retrovirus HERV-K, which we analyzed for a possible association with disease. We first investigated whether LTR segregate with DQ alleles in families. Members (n = 110) of 29 families with at least one diabetic child, unrelated patients with IDDM (n = 159), and healthy controls (n = 173) were analyzed. Genomic DNA was amplified for DQ LTR3 by a nested primer approach as well as for DQA1 and DQB1 second exons, to assign DQA1 and DQB1 alleles. DQ LTR segregated in 24 families along with DQ alleles. Of the 29 families, 20 index patients were positive for DQ LTR. The DQ LTR was in all patients on the haplotype carrying the DQA1 ^*0301 and DQB1 ^*0302 alleles. A majority of patients had DQ LTR (62%) compared with controls (38%) (p < 1.3 × 10^{− 5}), even after matching for the high-risk alleles DQA1 ^*0501, DQB1 ^*0201-DQA1 ^*0301, and DQB1 ^*0302 (79% of patients and 48% of controls; p < 0.02). Subtyping for DRB1 ^*04 alleles in all DQB1 ^*0302 ⁺ individuals showed 56% DRB1 ^*0401, DQB1 ^*0302 [LTR⁺ patients vs. 29% controls with the same haplotype (p < 0.002). In conclusion, these data demonstrate the segregation of DQ LTR with DQA1, DQB1 alleles on HLA haplotypes. Furthermore their presence on DRB1 ^*0401-, DQA1 ^*0301-, and DQB1 ^*0302-positive haplotypes suggest that they contribute to DQ-related susceptibility for IDDM. Human Immunology 50, 103–110 (1996)     

HLA-DRB1* and allergy to Parietaria: linkage and association analyses

In this study, we used the affected sibling-pairs approach to investigate the linkage of HLA (human leukocyte antigen)-DRB1* with phenotypes related to allergy to Parietaria, the most common pollinosis in Mediterranean countries. The study population consisted of 51 nuclear families (235 subjects). Linkage was detected with Parietaria skin test positivity (p < 0.01), presence of IgG and IgE antibodies specific for the major allergen Par o 1 (p < 0.020 and p < 0.025, respectively), and absence of Par o 1-specific IgE (p < 0.020).

High levels of Par o 1-specific IgG were associated with DRB1*1101 and/or DRB1*1104 (p < 0.0001 and p < 0.0119, respectively) in parents and probands. High levels of Par o 1-specific IgE were associated with DRB1*1104 in parents (p < 0.017) and with DRB1*1101 in probands (p < 0.0146). When siblings were categorized according to high/low total IgE levels (125 IU/ml and <125 IU/ml, respectively), high IgE antibody response was associated with DRB1*1104 in siblings with low total IgE (p < 0.034) and with DRB1*1101 in siblings with high total IgE (p < 0.05).

These results demonstrate that HLA-DRB1*, or genes in linkage disequilibrium, contributes to susceptibility to Parietaria allergy and that total IgE levels can discriminate population subsets where different alleles (at the HLA region or at loci in linkage disequilibrium) contribute to control allergen-specific IgE synthesis.     

HLA and systemic lupus erythematosus in Chinese

Seventy-five unrelated Chinese SLE patients were HLA typed and subdivided into mild and severe disease The HLA-B13 was associated with mild disease Fourteen of 30 (46.7%) mild disease patients had B13 compared to 63/330 (19.1%) normal subjects (p < 0 0005, corrected p < 0 013, RR = 3 7) The HLA-B17 on the other hand was observed in 29% of 45 severe disease patients compared to 13 9% of 330 normal subjects (p < 0 01, RR = 2.5). The frequency of HLA-B17 in 11 patients who died was even higher (45 5%).     

A female advantage in the imitation of gestures by preschool children

Adult research has suggested a sex difference in the neuroanatomical organization and efficiency of the limb praxis system. The present study used a videotape analysis of movement errors to investigate whether a sex difference in limb praxis can be identified in childhood. Preschool girls (n = 33) committed fewer “praxic” errors than boys (n = 31) (i.e., errors that resemble those seen in limb apraxia) when imitating non-representational gestures and when imitating common, everyday gestures with representational content. In none of the conditions did the female advantage generalize to “nonpraxic” error categories. The findings were not explained by a female advantage in cognitive development. The current study supports the hypothesis of a sex difference in limb praxis that is present from an early age.     

A high n-6 polyunsaturated fatty acid diet reduces muscarinic M2/M4 receptor binding in the rat brain

The aim of this study was to examine the influence of different fat diets on muscarinic acetylcholine receptor binding. Nineteen male Sprague–Dawley rats were divided into four groups and fed a diet of either high saturated fat, n-6 polyunsaturated fatty acid (PUFA), n-3 PUFA or low fat (control) for 8 weeks. Using quantitative autoradiography, [³H]pirenzepine binding to muscarinic M1/M4 receptors and [³H]AF-DX384 binding to M2/M4 receptors were measured throughout the brain in all four groups. The main findings were that compared to the low fat control group, M2/M4 receptor binding was significantly reduced in the dorsolateral, dorsomedial and ventromedial parts of the caudate putamen (61–64%, p < 0.05), anterior cingulate cortex (59%, p < 0.01), dentate gyrus and CA1–3 fields of the hippocampus (32–43%, p < 0.01) of rats on a high n-6 PUFA diet; however, no differences in M1/M4 receptor binding densities between the four groups were observed. These results suggest that a diet high in n-6 PUFA, but not of n-3 PUFAs or saturated fat, may selectively alter M2/M4 receptor-mediated signal transduction in the rat brain.     

Early effects of continuous low-dosage dl-norgestrel administered alone or with estrogen

Twenty-six postmenopausal women participated in a double-blind trial involving treatment according to a Latin square design with either (i) dl-norgestrel alone (0.075 mg/day) continuously for two cycles, (ii) estradiol-17β alone (1 mg on 25 of 28 days) for two cycles, or (iii) the combined hormones for six cycles. A placebo control cycle followed each hormonal treatment. Plasma triglycerides decreased by an average of 22% during treatment with either dl-norgestrel alone (123 ±11 vs. 160 ±10 mg/dl, n = 25, P < 0.005) or combination therapy (126 ±11 vs. 162 ±11, n = 25, P < 0.005) as compared with control. Plasma total cholesterol fell by 5% during two cycles of treatment with either dl-norgestrel alone (229 ±11 vs. 242 ±10 mg/dl, n = 25, P < 0.02) or combination therapy (233 ± 11 vs. 246 ±10, n = 25, P < 0.05) versus placebo. During the fifth and sixth cycles of combination therapy 94% of cycles were free of flushing (vs. 31% for control, P < 0.01), 64% of cycles were free of spotting not requiring protection (control 75%), 96% of cycles were free of vaginal bleeding (control 100%), endometrial biopsy showed inactive endometrium in nine of the 10 subjects re-biopsied, fasting blood pyruvate decreased by 20% (P < 0.05) and diastolic blood pressure fell by 4% compared with control (P < 0.05), whereas glucose tolerance was unchanged. There was a significant reduction in vasomotor flushing beginning with the third to fourth cycles of combination therapy.     

Brain Creatine Kinase with Aging in F-344 Rats: Analysis by Saturation Transfer Magnetic Resonance Spectroscopy

We measured in vivo forward flux of the creatine kinase reaction in rat forebrain in young (Y: 6 month, n = 13), mid-aged (M: 12 month, n = 7) and aged (O: 27 month, n = 10) animals using 31P magnetic resonance saturation transfer. Forward flux was reduced in the aged rats (Y: 0.42 ± 0.08; M: 0.41 ± 0.10; O: 0.31 ± 0.03 s⁻¹ ± SD; p = 0.008 O vs. Y). In vitro studies in a subset of the same rats showed a parallel decline in CK activity (Y: 2.16 ± 0.40; M: 2.17 ± 0.25; O: 1.56 ± 0.06 IU ±S.D.; p = 0.002 O vs. Y). The in vivo spectroscopic and in vitro biochemical measures were significantly correlated. Reduced creatine kinase activity could account for the observed decreased forward flux in aging brain. Intracellular pH, phosphocreatine/inorganic phosphate ratio, and phospocreatine/γ-adenosine triphosphate ratio did not differ between groups. Forward flux may represent a better measure of brain energy function than relative phosphocreatine or adenosine triphosphate levels observable in vivo.