C.J.A. de Groot

Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in Lewis rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) which causes paralysis. Several studies have reported the involvement of Ia antigen‐expressing cells in the pathogenesis of EAE. Interferon‐gamma (IFN‐γ) can induce Ia antigen expression on a wide range of cells. We examined the effect of IFN‐γ on EAE in Lewis rats. Systemically administered IFN‐γ did not change the disease course of EAE, whereas IFN‐γ applied locally into the ventricular system of the CNS resulted in complete suppression of clinical signs. Furthermore, we found that systemic administration of anti‐IFN‐γ just prior to the onset of clinical symptoms resulted in a more severe disease course. We conclude that IFN‐γ is capable of exerting a suppressive action in EAE, possibly through induction of Ia antigen expression or through the induction of suppressive mechanisms locally in the CNS.

Axonal damage in the spinal cord of MS patients occurs largely independent of T2 MRI lesions

Objective: To determine the degree of axonal damage in relationship to signal abnormalities on T2-weighted high-resolution MRI in spinal cord tissue of patients with MS. Methods: Spinal cord specimens of nine patients with MS and four controls were imaged at high resolution (4.7 T) in an axial plane and scored for lesions with increased signal intensity (SI). Histopathologic sections were cut and immunostained with NE14 (neurofilament marker) and Luxol fast blue (myelin stain). For each area, axonal density and diameter were quantified; axonal irregularity, NE14 axonal staining intensity, and myelin content were semiquantitatively scored. Included were 209 areas from MS cases and 109 areas from control cases distributed over lateral, posterior, and anterior columns. Results: In control cases, no SI changes were found, average density of axons was 26,989/mm2, average diameter was 1.1 μm, and all scores for axonal irregularity, NE14 staining intensity, and myelin were normal. In MS cases, areas with increased SI were found, average axonal density was 11,807/mm2 (p < 0.0001), and average axonal diameter 2.0 μm (p = 0.001). Areas with high SI on MRI had lowest axonal density (average count: 10,504/mm2; range: 3,433 to 26,325/mm2), largest diameter (average: 2.3 μm; range: 1.0 to 4.0 μm), and highest axonal irregularity and NE14 staining intensity compared to normal appearing cord tissue (NACT). However, NACT of MS cases also had lower axonal density (14,158/mm2) and higher average axonal diameter (1.6 μm) than controls. Conclusions: Marked axonal loss occurs in MS spinal cords, largely independent of the degree of signal abnormality on T2-weighted MRI.

Macrophages in the central nervous system of the rat

In an immunohistochemical study using monoclonal antibodies, which exclusively recognize cells of the monocyte-macrophage lineage, and monoclonal antibodies against the Ia-antigen, we describe the occurrence of macrophages in the developing and adult central nervous system (CNS). In normal adult brain, no macrophages could be detected in the CNS parenchyma; only in the meninges and the choroid plexes were a few macrophages found. During ontogeny, numerous phagocytic cells infiltrated the CNS parenchyma; these cells which did not express Ia are blood-borne. About three weeks after birth, all macrophages had disappeared from the CNS. As microglia in adult and developing brain do not stain with the anti-macrophage antibodies, we suggest that microglial cells are not related to the mononuclear phagocyte system and do not have a hematogenous origin.

Functional expression of CXCR3 in cultured mouse and human astrocytes and microglia

It has been established recently that inflammation of the CNS is accompanied by an expression of chemokines within the CNS. Several lines of evidence suggest that chemokines within the CNS initiate and orchestrate the infiltration of the inflamed brain by blood leukocytes. It is also known that endogenous cells of the CNS express functional chemokine receptors, raising the possibility that chemokines may be involved in intercellular signalling between brain cells during brain inflammation. It was shown recently that two chemokine ligands for CXCR3 are induced rapidly in damaged neurons. Little is known yet on the function of neuronal chemokine expression. In order to investigate whether neuronal chemokines contribute to endogenous signalling within the CNS we investigated possible expression of CXCR3 in glial cells. Reverse transcription-polymerase chain reaction experiments and in situ hybridization analysis showed that cultured astrocytes and microglia from both mouse and human sources express CXCR3 mRNA. Protein expression of CXCR3 in both cell types was detected by immunocytochemistry. Moreover, stimulation of cultured glial cells with chemokine ligands for CXCR3 induced intracellular calcium transients and chemotaxis, indicating the functional expression of CXCR3. These results indicate that glial cells in culture functionally express the chemokine receptor CXCR3. Since it has been shown that brain damage rapidly induces expression of neuronal chemokines that activate CXCR3, we suggest that glial CXCR3 might contribute to an intercellular signalling system in the CNS related to pathological conditions.

Sequential fractionation of sediment phosphate

By means of sequential extractions with Ca-NTA and EDTA, a separation was performed between Fe(OOH) ≈ P and CaC03≈P in a few sediments; the remaining fraction, considered to be organic phosphate, was quantified as well. We found that with the commonly used method of extraction with NaOH and H2S04, less Fe(OOH) ≈ P and much more CaC03 ≈ P was found than with the chelating extractants. The organic phosphate pool in live and dead algal material and in some mud samples was partly hydrolysed and therefore recovered as inorganic phosphates with classical extractions. The difference between chelating extractants and the classical ones is discussed.

Low-dose cyclosporin A induces relapsing remitting experimental allergic encephalomyelitis in the Lewis rat

Experimental allergic encephalomyelitis (EAE) in Lewis rats is an acute monophasic autoimmune disease. It can be treated prophylactically and therapeutically with high doses of cyclosporin A (CsA). Here we demonstrate that low-dose CsA does not prevent a first attack of EAE, but, on the contrary, induces a chronic relapsing form of the disease in 100% of Lewis rats examined. Possible explanations for the high relapse rate after low-dose CsA treatment are discussed. Further studies will be needed to evaluate the immunological mechanisms responsible for these results.

Reciprocal regulation of glutamine synthetase and carbamoylphosphate synthetase levels in rat liver

In glucocorticosteroid-treated diabetic rats, glutamine synthetase enzyme levels in the liver are decreased 3-fold, whereas carbamoylphosphate synthetase enzyme levels are increased 2.3-fold. In addition, immunohistochemistry shows that under these conditions the distribution of carbamoylphosphate synthetase is expanded over the entire liver acinus, whereas that of glutamine synthetase is reduced to very few cells bordering the central (terminal hepatic) veins. Using a newly isolated cDNA complementary to rat liver glutamine synthetase mRNA, we show that this regulation is primarily effected at a pretranslational level. (For data on carbamoylphosphate synthetase mRNA levels, see De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67). Furthermore, hybridization studies show stimulatory effects of both glucocorticosteroids and thyroid hormone on the glutamine synthetase mRNA level. Attempts to localize glutamine synthetase mRNA within the liver acinus by selective destruction of the pericentral zone failed because of generally low levels of liver mRNAs after CCl4 poisoning. In contrast to the situation after birth, significantly higher glutamine synthetase mRNA/enzyme activity ratios in fetal rat liver point to the presence of additional post-transcriptional control mechanisms before birth. These findings complement similar observations on carbamoylphosphate synthetase gene expression (De Groot et al. (1986) Biochim. Biophys. Acta 866, 61-67).

Reluctance to continue breastfeeding in The Netherlands.

A prospective cohort study of breastfeeding practice (0–4 mo) was carried out in well‐baby clinics. The cohort included 4438 newborns brought to a clinic for the first time between 1 April and 1 July 1998. The odds ratios of demographic and gestational variables connected with the start and duration of breastfeeding were measured. The frequency of the reasons why breastfeeding was interrupted was determined. At birth 71% of the infants included in this study were exclusively breastfed. After 4 mo the percentage had dropped to 21%. Breast milk was replaced directly by formula (23%) or by a mixture of breast milk and formula (77%). Exclusive breastfeeding was given irrespective of the mothers cultural background. Higher education appeared to be the most decisive factor for the initiation of exclusive breastfeeding; higher parity was found to be the most decisive factor for continuation. In 46% of cases the infants health and behaviour caused mothers to stop exclusive breastfeeding; in 38% the reasons were mother related; in 17% both mother‐ and infant‐related motives were mentioned.

Developmental and hormonal regulation of carbamoyl-phosphate synthase gene expression in rat liver: evidence for control mechanisms at different levels in the perinatal period

Carbamoyl-phosphate synthase gene expression is found to be primarily regulated by conditions that enhance hepatic glucocorticosteroid levels (hormone injections) and cyclic AMP levels (induction of diabetes). After birth, changes in the level of carbamoyl-phosphate synthase protein follow changes in the level of carbamoylphosphate synthase mRNA, suggesting a pretranslational control mechanism. In fetal rats, carbamoyl-phosphate synthase gene expression is regulated by the same factors as in adults. However, both the level to which carbamoyl-phosphate synthase mRNA can accumulate and the extent to which mRNA can be translated appear to be limited, indicating control mechanisms at the pretranslational and translational level. Finally, in the immediate postnatal period, a transient but pronounced decrease in the rate of degradation of carbamoyl-phosphate synthase protein may play a role in the accumulation of the enzyme.

Regulation of mRNA levels of rat liver carbamoylphosphate synthetase by glucocorticosteroids and cyclic AMP as estimated with a specific cDNA.

The construction and cloning of a cDNA complementary to the mRNA of rat liver carbamoylphosphate synthetase (ammonia) is described. Using this cDNA, the size of the mature, cytosolic carbamoylphosphate synthetase (ammonia) mRNA is estimated to be 6.0 Kb. The levels of carbamoylphosphate synthetase (ammonia) mRNA in liver are shown to be regulated by glucocorticosteroids and cyclic AMP. By studying mRNA levels of carbamoylphosphate synthetase, albumin and phosphoenolpyruvate carboxykinase, using specific cDNA clones, we show that carbamoylphosphate synthetase gene expression, like that of albumin is liver-specific.