Expression of immune-related molecules is downregulated in twitcher mice following bone marrow transplantation.

Twitcher (twi/twi) is a murine model of a human genetic demyelinating disease, globoid cell leukodystrophy (Krabbe disease). The affected mice usually die before reaching age 45 days, having demyelination associated with extensive glial activation. The twi/twi mice that receive wild-type bone marrow transplantation (BMT) survive up to 3 times longer with improved pathology. We hypothesize that immune-related molecules such as cytokines and chemokines are partly responsible for the demyelination in twi/twi, and that the decrease in the expression of such molecules following BMT contributes to clinico-pathological improvement. Cells expressing TNF-alpha, MCP-1, and MIP-1beta were conspicuous in the twi/twi CNS accompanied by infiltration of Ia+ and CD8+/CD3- hematogenous cells. These cells decreased gradually after BMT TNF-alpha mRNA and mRNA of C-C chemokine families, including MCP-1, IP-10, MIP-1alpha, MIP-1beta, and RANTES, were upregulated in the twi/twi CNS but downregulated gradually following BMT. In twi/twi that survived to 20 wk of age, cells expressing TNF-alpha, MCP-1, MIP-1beta, Ia, or CD8 were hardly detected and pathology was clearly improved. These results are consistent with the hypothesis that cytokine expression in glial cells contributes (to some extent) to the pathogenesis of demyelinating lesions in the twi/twi mice.

Absence of macrophage-inflammatory protein-1alpha delays central nervous system demyelination in the presence of an intact blood-brain barrier.

Chemokines are small chemotactic cytokines that modulate leukocyte recruitment and activation during inflammation. Here, we describe the role of macrophage inflammatory protein-1alpha (MIP-1alpha) during cuprizone intoxication, a model where demyelination of the CNS features a large accumulation of microglia/macrophage without T cell involvement or blood-brain barrier disruption. RNase protection assays showed that mRNA for numerous chemokines were up-regulated during cuprizone treatment in wild-type, C57BL/6 mice. RANTES, inflammatory protein-10, and monocyte chemoattractant protein-1 showed greatest expression with initiation of insult at 1-2 wk of treatment, whereas MIP-1alpha and beta increased later at 4-5 wk, coincident with peak demyelination and cellular accumulation. The function of MIP-1alpha during demyelination was tested in vivo by exposing MIP-1alpha knockout mice (MIP-1alpha(-/-)) to cuprizone and comparing pathology to wild-type mice. Demyelination at 3.5 wk of treatment was significantly decreased in MIP-1alpha(-/-) mice ( approximately 36% reduction), a result confirmed by morphology at the electron microscopic level. The delay in demyelination was correlated to apparent decreases in microglia/macrophage and astrocyte accumulation and in TNF-alpha protein levels. It was possible that larger effects of the MIP-1alpha deficiency were being masked by other redundant chemokines. Indeed, RNase protection assays revealed increased expression of several chemokine transcripts in both untreated and cuprizone-treated MIP-1alpha(-/-) mice. Nonetheless, despite this possible compensation, our studies show the importance of MIP-1alpha in demyelination in the CNS and highlight its effect, particularly on cellular recruitment and cytokine regulation.

Phagocytosis and clearance of apoptotic cells is mediated by MER.

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.

Falsely elevated INRs in warfarin-treated patients with the lupus anticoagulant.

The Lupus Anticoagulant (L.A.) is an antibody that prolongs the clotting time of in-vitro laboratory tests by binding phospholipid in the test system. Patients with the L.A. are at increased risk for development of venous and arterial thrombosis but not hemorrhage. Therefore, many patients with the L.A. are being treated with warfarin sodium to prevent reoccurrence of thrombosis. This oral anticoagulant therapy is traditionally regulated by periodic determination of the Prothrombin Time (PT). This test is usually unaffected by the L.A. However, we have recently identified a small series of patients with the L.A. in whom the PT is affected by the L.A. This interference is manifest as an artifactually increased International Normalized Ratio (INR). These patients were identified by failure to achieve significant correction of the PT with addition of an equal volume of normal plasma to the patient plasma and a Factor X level discordant with the PT INR Interference in determination of the PT by the L.A. was found to occur in 6.5% of patients identified with the L.A. by our laboratory. It is suggested that patients with this complication of anticoagulant therapy be monitored by measurement of Factor X levels rather than the PT INR. Failure to recognize this complication may result in inadequate anticoagulation and recurrent thrombosis.

Pooling blood donor samples to reduce the cost of HIV-1 antibody testing.

Accurate, low cost testing of donated blood is a goal of the global effort to reduce the spread of the human immunodeficiency virus (HIV-1). We describe a modified enzyme immunoassay (EIA) method for detecting HIV-1 antibody (anti-HIV-1) in 15-sample pools. In this preliminary study, the modified EIA was as sensitive for detecting weakly seropositive samples, and as specific for testing HIV-1 Western blot-negative or Western blot-indeterminate results, as testing individual samples by the standard EIA. A simulation of field operations was conducted using pools of blood donor samples collected in the United States and in Shanghai, People's Republic of China. Implementation of the modified EIA method and testing 15-sample pools for anti-HIV-1 is a reliable strategy for reducing the cost of large scale testing of donated blood for anti-HIV-1 in areas of low seroprevalence.

Denervation of primate extraocular muscle. A unique pattern of structural alterations.

Extraocular muscles differ from most other skeletal muscles in terms of constituent fiber types and innervation pattern. The rules that govern fiber responses to various experimental interventions for most skeletal muscles, therefore, may not strictly apply to the extraocular muscles. In this study, denervation of the extraocular muscles of Cynomolgous monkeys, Macaca fascicularis, was accomplished by intracranial transection of the oculomotor nerve. Survival times of 3-167 days were allowed, and muscles were processed for analysis by light and electron microscopy. Short-term alterations involved all muscle fiber types and included retraction of neuromuscular junctions, mild myofibril disruption and inflammatory cell infiltration. Long-term morphopathological changes were most apparent in the orbital singly innervated fiber type and its global layer counterpart. These alterations consisted of dispersion of the mitochondrial aggregates which characterize this fiber type. Only occasional fibers (all types) exhibited severe vacuolar atrophy or myofilament breakdown despite the occurrence of only limited reinnervation. When extensive reinnervation did occur, the characteristic layered organization of the extraocular muscles was preserved and fiber type grouping was not apparent. Taken together, these findings indicate that the extraocular muscles exhibit a resilience to denervation beyond that observed for other skeletal musculature.

Extraocular myotoxicity of the retrobulbar anesthetic bupivacaine hydrochloride.

Morphopathological changes induced in the extraocular muscles by the local anesthetic agent bupivacaine hydrochloride were studied in the monkey using light and electron microscopy. Retrobulbar anesthetic blocks, using 0.75% bupivacaine hydrochloride, were performed in five adult cynomolgus monkeys. Morphological alterations in extraocular muscle fiber types were examined following survival periods of 3-27 days. Bupivacaine injections produced a mild and very limited myopathic response, with changes largely restricted to the global layer singly-innervated muscle fiber type which is characterized by low mitochondrial content. For survival times beyond 3 days, this fiber type exhibited peripheral migration and swelling of mitochondria and an outside-in pattern of myofibril dissolution. Some affected fibers also exhibited the Ringbinden or ring fiber pathology. Maximal myotoxic response was observed at 14 days after injections, and pathological changes were largely resolved by 27 days. A more limited analysis of the effects of retrobulbar injection of lidocaine revealed similar morphopathological responses, thereby suggesting that these effects are a property common to the entire class of aminoacyl anesthetics. In contrast to previous observations in other skeletal musculature, the extraocular muscles proved to be unexpectedly resistant to local anesthetic treatment as only limited alterations were observed. The observed muscle fiber type specificity was interpreted to result from differences in the relative ability of muscle fiber types to adapt to anesthetic-induced elevations of intracellular free calcium levels.