Changes in cannabinoid CB(1) receptor functionality in the female rat prefrontal cortex following a high fat diet.

AIMS: A high fat diet (HFD) has been found to affect neurotransmission in the prefrontal cortex, but the effects of this dietary regime upon the endocannabinoid system has not been studied in this brain region. In consequence, in the present study, we have investigated the effect of HFD for up to 20 weeks upon the endocannabinoid system in the prefrontal cortex of female rats. MAIN METHODS: CB1 receptor functionality was measured using CP55,940-stimulated [(35)S]GTPγS autoradiography. Fatty acid amide hydrolase and monoacylglycerol lipase activities were analysed in brain regions by assessing rates of [(3)H]anandamide and JZL184-sensitive [(3)H]2-oleoylglycerol hydrolysis, respectively. KEY FINDINGS: In the prefrontal cortex, a significantly greater stimulation of [(35)S]GTPγS binding by CP55,940 was seen following 4-12, but not 16-20 weeks of HFD. No significant changes were seen for the caudate-putamen, CA1-CA3 region of the hippocampus or the dentate gyrus. The increased response for the 12 week animals was not accompanied by a significant change in the receptor density, measured with [(3)H]CP55,940 autoradiography. No significant changes in the activity of the endocannabinoid hydrolytic enzymes fatty acid amide or monoacylglycerol lipase were seen in the prefrontal cortex, hippocampus, amygdala or hypothalamus following either 12 or 20 weeks of HFD. SIGNIFICANCE: It is concluded that HFD produces an increased CB1 receptor functionality in the prefrontal cortex of female rats. Given that the endocannabinoid system regulates neurotransmission in the prefrontal cortex, the present data would implicate this system in the disturbed prefrontal cortical activity in this region following a high fat diet.

Altered VH6-D-JH repertoire in human insulin-dependent diabetes mellitus and autoimmune idiopathic thrombocytopenic purpura.

We have characterized the peripheral B cell repertoire in T cell-mediated insulin-dependent diabetes mellitus (IDMM) and in B cell-mediated autoimmune idiopathic thrombocytopenic purpura (AITP). The VH6-containing repertoire in adult patients with IDDM or AITP and healthy control subjects was investigated by PCR amplification using VH6- and JH-specific primers. Nucleotide sequence analysis of VH6-D-JH rearrangements showed an abnormally high frequency of somatic mutations in non-functional rearrangements from diabetic (3. 58 %) as well as AITP patients (3.18 %), compared to controls (0.4 % and 1.43 %, respectively; p < 0.05). In contrast, the mutation frequency among functional rearrangements was 2.4 - 3 times lower in patients compared to controls ( p < 0.05). Detailed analysis of the VH6 genes carrying mutations showed that the underlying mechanism for this observation is probably different for the two diseases. Analysis of D- and JH gene usage revealed additional deviations from the normal pattern. Taken together, these results suggest defects in the mechanisms controlling selection of the B cell repertoire in patients with IDDM or AITP.

Age-related impaired affinity maturation and differential D-JH gene usage in human VH6-expressing B lymphocytes from healthy individuals.

To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we analyzed a panel of 179 polymerase chain reaction-derived VH6-D-JH rearrangements from cord blood, peripheral blood, and spleen. Nucleotide sequence analysis of the CDR3 region shows that there is a difference in D and JH gene usage in functional rearrangements between lymphocytes from peripheral blood and spleen. Analysis of the VH6 gene shows that the mutational frequencies rise from 0.81% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decrease to 0.80% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20-49 years, and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. These data show an age-related impaired affinity maturation which might relate to the decrease in immunological responsiveness among the elderly.

Aberrant V(H) gene utilization in patients with established insulin dependent diabetes mellitus.

We have compared the B-lymphocyte repertoire in seven IDDM patients with 12 healthy controls by examining the variable heavy (V(H)) gene expression. The V(H) gene representation in the pool of pokeweed mitogen (PWM) stimulated, immunocompetent B cells and in the pool of naturally activated plasma cells (actual repertoire) was analysed by RNA-RNA in situ hybridization. Differences between IDDM patients and normal controls in the relative expression of several V(H) gene families were observed. In IDDM patients, the V(H)3 was significantly underrepresented in the PWM stimulated repertoire. In the actual B cell repertoire the V(H)5 clones were underrepresented among diabetic patients. Moreover, the altered distribution of V(H) gene usage between the PWM stimulated repertoire and the actual repertoire observed in normal controls was found to be less pronounced in the IDDM patients. This observation suggests a defect in the V-gene directed cellular selection occurring between resting, immunocompetent B cells and naturally activated plasma cells. The possible implication of the observed aberrations in the B cell selection process for the pathogenesis of autoimmunity is discussed.

Establishment and characterization of RAG-2 deficient non-obese diabetic mice.

The authors have established a new immunodeficient mouse strain on the genetic background of the diabetes prone non-obese diabetic (NOD) mouse. A deletion mutant of the RAG-2 gene was back crossed 10 generations onto the NOD/Bom strain background. The homozygous NODrag-2-/- mice lack functionally mature B and T lymphocytes and do not develop insulitis or diabetes throughout life. In contrast, heterozygous NODrag-2+/- develop both insulitis and diabetes with an incidence similar to the wild type NOD mice. In transfer experiments, spleen cells from diabetic NOD donors were found to transfer disease to NODrag-2-/- recipients similar to what has been previously observed in transfer to irradiated NOD recipients or to immunodeficient NOD-scid/scid mice. While resembling the recently established NOD-scid/scid mice in many respects, the NODrag-2-/- mice represents an advantageous model for reconstitution of the pathogenesis of murine IDDM as it does not produce any endogenous, mature T or B lymphocytes.