Mapping the rat somatosensory pathway from the anterior cruciate ligament nerve endings to the cerebrum

The anterior cruciate ligament (ACL) serves as the primary restraint to anterior tibial translation. In addition to this biomechanical function, the ACL appears to have a function in neuromuscular control. This hypothesis was formulated after the discovery of mechanoreceptors within the ACL. The full somatosensory pathway from the ACL to the cerebrum has yet to be elucidated. In order to map this sensory pathway, we conducted a viral trans-synaptic tracing experiment using the neurotropic pseudorabies virus (PRV). The pseudorabies virus was injected into the ACL of rats and allowed to replicate and spread trans-synaptically for 6-7 days. The brain and spinal cord of each sacrificed rat was then removed and processed immunohistochemically to detect the presence of PRV. PRV-immunoreactive neurons were found to be localized in several different regions from the spinal cord to the cerebrum. Four nuclei in the reticular formation of the brain stem demonstrated strong positive labeling: the mesencephalic reticular nucleus, magnocellular reticular nucleus, paragigantocellular reticular nucleus, and gigantocellular reticular nucleus. This finding suggests that the nerve endings of the rat ACL project into the cerebrum and that the reticular formation may play an important role in the afferent pathway of those nerve endings.     

The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.     

The role of major and minor histocompatibility antigens in active enhancement of rat kidney allograft survival by blood transfusion

Rats given a blood transfusion do not reject a subsequent kidney allograft from the same donor strain. This effect is strain-specific so that pretransplant blood transfusion from a LEW (RT1l) rat will protect a LEW kidney in a DA (RT1a) recipient but not a PVG/c (RT1c) kidney. The same is true in other combinations. Using DA or PVG.RT1a congenic recipients we have shown that sharing of all or part of the MHC or of minor alloantigens by the blood transfusion and kidney transplant donors is sufficient to prolong allograft survival. Activation of suppression against minor alloantigens appears to require two signals: one is the minor alloantigen and the other may be a major histocompatibility complex alloantigen.     

Cyclic AMP inhibits macrophage suppressor function and enhances lymphocyte proliferation.

The effects of increasing the level of cyclic AMP (cAMP) activity on lymphocyte proliferation in the rat mixed lymphocyte reaction (MLR) were investigated. Dibutyryl cAMP (dbcAMP) and prostaglandin E2 (PGE2) produced a dose-dependent reduction in proliferation in the lymph node (LN) MLR, but produced a substantial increase in proliferation in the spleen MLR at the lower concentrations used (10(-5)-10(-4) M dbcAMP; 10(-7)-10(-6) M PGE2). Enhancement of proliferation was dependent on the presence of macrophages and was probably due to inhibition of macrophage activation. This was based on the following findings: (1) spleen MLR proliferation was lower than that in the LN MLR; (2) depletion of spleen macrophages increased proliferation in the spleen MLR and addition of these macrophages to the LN MLR reduced proliferation; (3) macrophage depletion from the spleen MLR abolished the proliferation-enhancing effect of dbcAMP. In conclusion, cAMP enhances lymphocyte proliferation in this system, apparently as a consequence of suppressing the inhibitory influence of macrophages.     

Environmental education

The need for a new profession devoted to environmental matters is asserted. The qualities of such a profession are sketched, and it is argued that new initiatives in environmental education are needed in the form of graduate, professional programs with primary emphasis on practice. An example 2-year program is presented. A fundamental requirement is scientific competence; undergraduate preparation in the sciences or engineering is mandatory. The graduate curriculum itself is built on three primary cores: environmental science and engineering, business and management, and public policy. Additionally, an environmental round table is proposed as a focal point for academic, industrial, governmental, and public discussion on environmental matters. The round table would provide oversight for the professional educational program and an affiliated research institute.     

Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. While its etiology is not well understood, genetic factors are clearly involved. Until recently, most genetic studies in MS have been association studies using the case-control design testing specific candidate genes and studying only sporadic cases. The only consistently replicated finding has been an association with the HLA-DR2 allele within the major histocompatibility complex (MHC) on chromosome 6. Using the genetic linkage design, however, evidence for and against linkage of the MHC to MS has been found, fostering suggestions that sporadic and familial MS have different etiologies. Most recently, two of four genomic screens demonstrated linkage to the MHC, although specific allelic associations were not tested. Here, a dataset of 98 multiplex families was studied to test for an association to the HLA-DR2 allele in familial MS and to determine if genetic linkage to the MHC was due solely to such an association. Three highly polymorphic markers (HLA-DR, D6S273 and TNFbeta) in the MHC demonstrated strong genetic linkage (parametric lod scores of 4.60, 2.20 and 1.24, respectively) and a specific association with the HLA-DR2 allele was confirmed (TDT; P < 0.001). Stratifying the results by HLA-DR2 status showed that the linkage results were limited to families segregating HLA-DR2 alleles. These results demonstrate that genetic linkage to the MHC can be explained by the HLA-DR2 allelic association. They also indicate that sporadic and familial MS share a common genetic susceptibility. In addition, preliminary calculations suggest that the MHC explains between 17 and 62% of the genetic etiology of MS. This heterogeneity is also supported by the minority of families showing no linkage or association with loci within the MHC.      

Self-help materials for anxiety: a randomized controlled trial in general practice.

The efficacy of a self-help package in treating chronic anxiety was evaluated in a randomized controlled trial in which the intervention group received self-help materials in the form of an audiotape and booklet, in addition to their current treatment. The intervention was successful in terms of mean depression scores (P = 0.01), anxiety scores (P = 0.04) and general health questionnaire scores (P = 0.02) which were significantly lower for the intervention group than for the controls. In addition, the depression scores fell faster for the intervention group than for the controls. The overall mean reduction in three months in adjusted depression scores was approximately two points greater for the intervention group than for the controls (P = 0.02). Clinicians welcomed the package as a valuable addition to the therapies available for managing chronic anxiety problems. Further studies should include larger sample sizes, taking into account the non-response to postal questionnaires over time.     

Enhancement of cALL immunogenicity by co-culture with a CD154 expressing 293 cell line

Pre-B cell acute lymphoblastic leukaemia (cALL) commonly occurs in young patients and although successful conventional therapies are available (such as cytotoxic drugs and bone marrow transplantation) for a proportion of patients (approximately 30%) these are ultimately unsuccessful. Recurrence of disease is a result of the failure of the immune system to recognize these abnormal cells and down-regulation of crucial molecules required for cognate CD4(+) T cell recognition has been postulated as a means of immune escape. In this study we show that an embryonic kidney cell line (293 cells) transfected with CD154 (40 L.1) are capable of not only maintaining the viability of primary ALL cells in culture but can also up-regulate the expression of a number of crucial molecules involved in antigen recognition. We show that 40 L.1 cell stimulation of primary ALL cell cultures can not only enhance the allogeneic and autologous MLR response to such cells but will also induce CTL effectors which are capable of lysing wild-type autologous ALL cells. It is therefore conceivable that such an approach could be used to generate an active anti-tumour response in patients, following conventional therapy, reducing the incidence of recurrence.