Fiberoptic microindentation technique for early osteoarthritis diagnosis: an in vitro study on human cartilage.

In this study, the capability of a fiber optic microindenter sensor to discriminate between healthy and slightly degenerated human articular cartilage samples is demonstrated. The purely optical indenter sensor is characterized by extremely reduced dimensions (0.125 mm in diameter and 27 mm in length) in comparison to existing indenter probes offering advantages for endoscopic deployment. The indenter sensor is intended to assist the surgeon in the identification of damaged articular cartilage. From each of seven specimens of human tibia plateau three samples showing different Outerbridge grading were extracted. On each sample stress-relaxation measurements were performed with eight indentation steps, each step being 40 µm and the relaxation of the material was observed for 240 s after each step. A viscoelastic model was used to fit the relaxation and to extract the characteristic parameters according to the model. A highly significant difference in stiffness (p value <0.01) was observed between the native (grade 0) and early diseased (grade 1) human cartilage samples demonstrating the potential of the fiber optic indenter for the diagnosis of cartilage breakdown.

Evidence for a motor and a non-motor domain in the human dentate nucleus--an fMRI study.

Dum and Strick (J. Neurophysiol. 2003; 89, 634-639) proposed a division of the cerebellar dentate nucleus into a "motor" and "non-motor" area based on anatomical data in the monkey. We asked the question whether motor and non-motor domains of the dentate can be found in humans using functional magnetic resonance imaging (fMRI). Therefore dentate activation was compared in motor and cognitive tasks. Young, healthy participants were tested in a 1.5 T MRI scanner. Data from 13 participants were included in the final analysis. A block design was used for the experimental conditions. Finger tapping of different complexities served as motor tasks, while cognitive testing included a verbal working memory and a visuospatial task. To further confirm motor-related dentate activation, a simple finger movement task was tested in a supplementary experiment using ultra-highfield (7 T) fMRI in 23 participants. For image processing, a recently developed region of interest (ROI) driven normalization method of the deep cerebellar nuclei was used. Dorso-rostral dentate nucleus activation was associated with motor function, whereas cognitive tasks led to prominent activation of the caudal nucleus. The visuospatial task evoked activity bilaterally in the caudal dentate nucleus, whereas verbal working memory led to activation predominantly in the right caudal dentate. These findings are consistent with Dum and Strick's anatomical findings in the monkey.

Lethal exacerbation of Pneumocystis carinii pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice.

Mice homozygous for the mutant allele scid (severe combined immunodeficiency) have been described as excellent models for Pneumocystis carinii (Pc) pneumonia (PCP), a major health problem in patients with acquired immune deficiency syndrome (AIDS) and other immunodeficiency states. Other microorganisms have been shown to infect AIDS patients simultaneously with Pc, but whether one opportunist is able to directly influence the pathogenicity of another has not been determined previously. We have deliberately coinfected scid mice (with extent Pc infection) with a variety of primarily pneumotropic viruses and bacteria and have identified pneumonia virus of mice as causing a dramatic increase in the density of Pc organisms and the morbidity due to PCP in immunodeficient scid mice. This finding has clinical significance in the management of PCP, in that the identification and treatment of coinfecting pneumotropic pathogens may be as important as treatment targeted at Pc. A search for other synergistic (or antagonistic) microorganisms and determination of their mechanism(s) of action in altering the progression of PCP is indicated.

A new mutation, gld, that produces lymphoproliferation and autoimmunity in C3H/HeJ mice.

A newly discovered autosomal recessive mutation, generalized lymphoproliferative disease (gld), in the C3H/HeJ strain of mice, determines the development of early onset massive lymphoid hyperplasia with autoimmunity. Significant lymph node enlargement is apparent as early as 12 wk of age. By 20 wk, lymph nodes are 50-fold heavier than those of coisogenic C3H/HeJ-+/+ mice. There is a concomitant increase in the numbers of peripheral blood lymphocytes. Analysis of C3H-gld lymph node lymphocyte subsets by immunofluorescence indicates an increase in numbers of B cells, T cells, and null (Thy-1-, sIg-) lymphocytes by 6-, 15-, and 33-fold compared with congeneic control mice. Serologically, gld/gld mice develop antinuclear antibodies (including anti-dsDNA), thymocyte-binding autoantibody, and hypergammaglobulinemia with major increases in several immunoglobulin isotypes. Mutant gld mice live only one-half as long as normal controls (12 and 23 mo, respectively). Interstitial pneumonitis was found in virtually all C3H-gld mice autopsied when moribund. Although immune complexes were detected in the glomerulus by immunofluorescence techniques, only 14% of the autopsied mice had significant lupus-like nephritis. Vascular disease was not found. The pattern of early onset massive lymph node enlargement, hypergammaglobulinemia, and production of antinuclear autoantibodies resembles the basic abnormal phenotype induced by the lpr (lymphoproliferation) mutation. The mutations gld and lpr are not allelic. Linkage studies indicate that gld is located between Pep-3 and Lp on chromosome 1. This new mutation adds another genetically well-defined model to the list of murine lymphoproliferative/autoimmune disorders that may be exploited to gain a clearer understanding of immunoregulatory defects and for identifying common pathogenetic factors involved in systemic autoimmune diseases.

Differences defined by bone marrow transplantation suggest that lpr and gld are mutations of genes encoding an interacting pair of molecules.

Homozygosity for either of the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations of mice causes the development of systemic lupus erythematosus-like autoimmune syndromes that are characterized by severe lymphadenopathy and highly elevated serum immunoglobulin levels. Although the mutations are nonallelic, analysis of homozygous lpr/lpr and gld/gld mice on the same strain background has indicated that the pathology and severity of the autoimmune syndromes induced by these mutations are indistinguishable. To explain this, it has previously been suggested that lpr and gld may represent mutations in molecules involved in sequential steps of an intracellular metabolic pathway of T cells. We have now investigated the behavior of both lpr and gld in a variety of bone marrow chimeras and have found that functional differences between lpr and gld become apparent after bone marrow transfer. Transfer of lpr/lpr bone marrow to irradiated congenic +/+ recipients caused the development of a graft-vs.-host-like lymphoid wasting syndrome, whereas transfer of gld/gld bone marrow to +/+ recipients resulted in development of a gld-like autoimmune syndrome. Additionally, gld/gld hosts behaved like +/+ hosts irrespective of the genotype of the donor bone marrow, whereas lpr/lpr hosts behaved unlike +/+ hosts when reconstituted with either lpr/lpr, gld/gld, or +/+ bone marrow. These are the first clear differences between these two mutations yet described. Our studies indicate that the molecule altered by the gld mutation is expressed only by bone marrow-derived cells, whereas the molecule altered by the lpr mutation is expressed by both bone marrow-derived cells and by one or more peripheral radioresistant cell populations. To reconcile these differences with the fact that homozygous lpr/lpr and gld/gld mice are indistinguishable, we suggest an alternative model for the relationship between the lpr and gld mutations in which the two molecules affected represent an interacting ligand-receptor pair expressed by different cells.

Abnormalities induced by the mutant gene, lpr. Patterns of disease and expression of murine leukemia viruses in SJL/J mice homozygous and heterozygous for lpr.

SJL/J mice heterozygous or homozygous for the lpr mutation were compared with SJL/J-+/+ mice for longevity, histopathology, antigenic characteristics of lymphocytes and expression of murine leukemia viruses (MuLV). In comparison to +/+ mice, lpr homozygotes had a markedly shortened life span, died with infiltrative pulmonary disease, but little or no renal disease, and expressed high levels of infectious ecotropic MuLV in lymphoid tissues. SJL-lpr/+ mice had a life span intermediate between SJL-+/+ and -lpr/lpr mice, died with lymphomas that histologically resembled the neoplasms of +/+ mice, and sometimes expressed high levels of ecotropic MuLV. The lymphomas of lpr/+ could be transplanted to +/+ recipients in 78% of cases, and continuous in vitro lines were established from some of them. Similar effects on virus expression or lymphoma development were not observed in other strains homozygous or heterozygous for the lpr mutation. These results indicate that the diseases expressed by mice homozygous for the lpr mutation are highly strain-dependent, and that this gene can have an effect in the heterozygous state in SJL mice.

Deficient interleukin 2 activity in MRL/Mp and C57BL/6J mice bearing the lpr gene.

Spleen cells from MRL-lpr and B6-lpr mice have a marked defect in the ability to produce interleukin 2 (IL-2) in response to concanavalin A stimulation. This defect precedes the onset of clinical illness, increases with age, and eventually becomes virtually absolute. It is not due to cellular suppression of IL-2 production, nor does it reflect the presence of a soluble inhibitor of IL-2 activity. Failure to restore IL-2 production with macrophage-replacing factors, such as interleukin 1 and phorbol myristic acetate, suggests that IL-2 deficiency reflects a primary T cell defect rather than a macrophage defect. MRL-lpr and B6-lpr spleen cells also have an age-dependent reduction in IL-2 response that apparently results from a deficiency of cell surface receptors for IL-2. Congenic MRL-+/+ and B6-+/+ mice, which lack the lpr gene responsible for accelerated autoimmunity and lymphoproliferation, have normal IL-2 activity. These findings suggest that a defect in IL-2 activity may contribute to impaired immunoregulation in mice bearing the lpr gene. The absence of such a defect in MRL-+/+ and B6-+/+ mice further suggests that a single autosomal recessive gene is responsible for IL-2 deficiency.

Spontaneous murine lupus-like syndromes. Clinical and immunopathological manifestations in several strains.

MRL/1 and BXSB male mice have a systemic lupus erythematosus (SLE)-like disease similar to but more acute than that occurring in NZB X W mice. The common elements of lymphoid hyperplasia, B-cell hyperactivity, autoantibodies, circulating immune complex (IC), complement consumption, IC glomerulonephritis with gp70 deposition, and thymic atrophy were found in all three kinds of SLE mice. On the basis of these common elements, SLE seen in these mice can be considered a single disease in the same sense that human SLE is one disease. The differences in the SLE expressed in the different mice are no greater than those found in an unselected series of humans with SLE. However, the significant quantitative and qualitative variations in abnormal immunologic expression suggest that different constellations of factors, genetic and/or pathophysiologic, may operate in the three murine strains and that each constellation is capable of leading, via its particular abnormal immunologic consequences, to the activation of common immunopathologic effector mechanisms that cause quite similar SLE-like syndromes. From an experimental point of view, the availability of several inbred murine strains of commonplace histocompatibility types that express an SLE-like syndrome makes possible innumerable manipulations which should help to elucidate the nature and cause(s) of this disorder.

Discrimination between healthy and degenerated bovine articular cartilage with a fiber Bragg grating based microindenter.

OBJECTIVE: In this study we aim to show that an optical fiber Bragg grating-based microindentation system, which has the potential to be deployed arthroscopically, can differentiate between healthy and degenerated articular cartilage, which represents an important challenge in minimally-invasive surgery. DESIGN: Twenty bovine osteochondral cylinders, extracted from the patellar groove of ten 24 months old animals were subjected to stepwise in vitro stress-relaxation indentation measurements. The indentation procedure comprised 15 indentation steps of 20 µm each, reaching a total depth of 300 µm. Ten samples remained untreated and served as a control group for healthy cartilage. A second group of ten samples was treated for 12 h with an aqueous trypsin solution (concentration 2.5%) to deplete the proteoglycans. For both groups and all indentation depths deeper than 100 µm, the step response functions of a two elements Maxwell-Wiechert model fitted well to the measured relaxation curves. RESULTS: The standard deviations of the identified stiffness parameters within each group were much smaller than the difference of the average stiffness values between both groups. Based on the measured stiffness values, the system was capable to discriminate between healthy and degenerated cartilage with a high level of significance (p < 0.001). The experimental results are also discussed in terms of the biomechanical changes of cartilage under the action of trypsin. CONCLUSION: The fiber Bragg grating microindentation system showed the capability to differentiate intact and proteoglycan depleted cartilage with high significance.

Both immunity and hyperresponsiveness to Pneumocystis carinii result from transfer of CD4+ but not CD8+ T cells into severe combined immunodeficiency mice.

The opportunistic pathogen Pneumocystis carinii (Pc) is considered to be the leading cause of morbidity in patients with AIDS. It is important, therefore, to determine the immunological mechanisms of resistance to Pc. We have taken advantage of the lack of both T and B lymphocytes in severe combined immunodeficiency (scid) mice to determine the critical factors in resistance to spontaneously acquired Pc pneumonia. Using adoptive transfer of unfractionated or fractionated lymphocyte subsets or hyperimmune serum from congenic normal donors, we have demonstrated that effective immunity to Pc results from the action of CD4+ but not CD8+ T cells (in the absence of antibody) or from humoral immunity (in the absence of T cells). However, responses of CD4+ T cells (but not antibody) to already well-established burdens of Pc are often accompanied by a fatal hyperinflammatory reaction. The activity of CD4+ T cells against Pc thus illustrates a broadly applicable principle that T cell immunity represents a critical balance between consequences beneficial and harmful to the host.

Multiple mechanisms of tumorigenesis in E mu-myc transgenic mice.

Transgenic mice bearing a c-myc oncogene under control of the immunoglobulin heavy chain enhancer (E mu-myc mice) reproducibly develop and die from tumors of the B lymphocyte lineage (J.M. Adams, A.W. Harris, C.A. Pinkert, L.M. Corcoran, W.S. Alexander, S. Cory, R.D. Palmiter, and R.L. Brinster, Nature (Lond.), 318: 533-538, 1985; W.Y. Langdon, A. W. Harris, S. Cory, and J.M. Adams, Cell 47: 11-18, 1986; A.W. Harris, C.A. Pinkert, M. Crawford, W.Y. Langdon, R.L. Brinster, and J.M. Adams, J. Exp. Med., 167: 353-371, 1988; reviewed in S. Cory and J.M. Adams, Annu. Rev. Immunol., 6: 25-48, 1988). Analysis of lymphocytes obtained by serial sampling of peripheral blood from individual hemizygous (E mu-myc/0) and homozygous (E mu-myc/E mu-myc) transgenic mice indicates that proliferation in the original host and transplantability into histocompatible recipients are distinct properties that can be acquired independently and in either order. These two types of transgenic mice differ in that homozygous mice have about one-fourth the life span of hemizygous mice and develop polyclonal, non-transplantable tumors in comparison to the oligoclonal, highly transplantable malignancies seen in hemizygous animals. In conclusion, the overall concept of malignancy is best viewed as an aggregate of the separable parameters of cellular proliferation, clonality, tissue invasiveness, metastasis, and (experimental) transplantability. The E mu-myc transgenic mouse represents an attractive model in which to investigate the multistep nature and alternative pathways of tumorigenesis.

Kinetic and organ-specific patterns of cytokine expression in acute graft-versus-host disease.

Although many cytokines have been previously implicated in graft-versus-host disease (GVHD), no study to date has comprehensively evaluated their expression over time or in different tissues affected by GVHD. Using a semi-quantitative reverse transcriptase-PCR technique and a murine model of acute GVHD, we have evaluated the expression levels of mRNA for a wide range of cytokines in spleen, gut and liver tissues at weekly intervals after bone marrow transfer. The earliest cytokine responses seen were increases in IL-2, IL-10, IFN-gamma, MIP-1 alpha and TNF-alpha in the spleen, suggesting a primarily Th1 pathway. Other cytokines (IL-1 alpha, IL-10 and MIP-1 alpha) were persistently elevated in GVHD mice, but were variable depending on the tissue. These data demonstrate that a wide range of cytokines are involved in the GVHD response and that their kinetic pattern of expression is different in various affected tissues.

Acyl-CoA binding proteins: multiplicity and function.

The physiological role of long-chain fatty acyl-CoA is thought to be primarily in intermediary metabolism of fatty acids. However, recent data show that nM to microM levels of these lipophilic molecules are potent regulators of cell functions in vitro. Although long-chain fatty acyl-CoA are present at several hundred microM concentration in the cell, very little long-chain fatty acyl-CoA actually exists as free or unbound molecules, but rather is bound with high affinity to membrane lipids and/or proteins. Recently, there is growing awareness that cytosol contains nonenzymatic proteins also capable of binding long-chain fatty acyl-CoA with high affinity. Although the identity of the cytosolic long-chain fatty acyl-CoA binding protein(s) has been the subject of some controversy, there is growing evidence that several diverse nonenzymatic cytosolic proteins will bind long-chain fatty acyl-CoA. Not only does acyl-CoA binding protein specifically bind medium and long-chain fatty acyl-CoA (LCFA-CoA), but ubiquitous proteins with multiple ligand specificities such as the fatty acid binding proteins and sterol carrier protein-2 also bind LCFA-CoA with high affinity. The potential of these acyl-CoA binding proteins to influence the level of free LCFA-CoA and thereby the amount of LCFA-CoA bound to regulatory sites in proteins and enzymes is only now being examined in detail. The purpose of this article is to explore the identity, nature, function, and pathobiology of these fascinating newly discovered long-chain fatty acyl-CoA binding proteins. The relative contributions of these three different protein families to LCFA-CoA utilization and/or regulation of cellular activities are the focus of new directions in this field.

Single and combined humoral and cell-mediated immunotherapy of Pneumocystis carinii pneumonia in immunodeficient scid mice.

Homozygous mutant scid/scid (severe combined immunodeficiency) mice (referred to as scid mice) lack both specific humoral and cell-mediated immune functions and are exemplary in vivo models for analysis of host-parasite relationships. In our colony, scid mice routinely and predictably develop spontaneous Pneumocystis carinii pneumonia (PCP) with high morbidity. Previous studies have identified both T cells (specifically, CD4+ cells) and antibody as independent mechanisms of effective anti-P. carinii resistance; however, CD4+ T cells also cause an often fatal hyperinflammatory reaction. The current study has explored the optimal application of these immune components for conferring protection against P. carinii. Anti-P. carinii hyperimmune serum was highly effective at reducing the number of P. carinii organisms in early, intermediate, and advanced stages of PCP and was capable of increasing the mean life expectancy of P. carinii-infected scid mice by more than threefold if provided on a continuing basis. When a short course of hyperimmune-serum therapy was provided prior to transfer of P. carinii-sensitized normal lymphocytes, scid mice were rendered permanently free of P. carinii without the pathological sequelae of the hyperinflammatory reaction. These findings are discussed in the contexts of mechanism and clinical relevance.

A Y chromosome associated factor in strain BXSB producing accelerated autoimmunity and lymphoproliferation.

Strain BXSB/Mp mice develop a spontaneous lupus-like syndrome which is strikingly accelerated in males. The accelerated autoimmune disease occurs in male F1 hybrids with strains NZB/BINJ, SJL/J, and C57BL/6J when the male parent is BXSB but not in the reciprocal hybrid male nor in females. The pattern is similar in F2 hybrids with strains NZB and SJL. The accelerated disease in males occurs only when the Y chromosome is derived from recombinant inbred strain BXSB and ultimately from strain SB/Le.

Interaction of mutant lpr gene with background strain influences renal disease.

The mutant gene lpr on the MRL/Mp strain of mice is responsible for converting a late onset glomerulonephritis into an early, aggressive, and fatal renal disease. This gene induces the proliferation of a unique subset of lymphocytes, the production of a variety of autoantibodies and shortened survival in MRL/Mp as well as in the genetically distinct strains C3H/HeJ, C57BL/6J, and AKR/J. The present study examined in detail the role of the lpr gene in the formation of lupus nephritis. The results show that C3H-lpr and B6-lpr mice do not develop nephritis while the AKR-lpr strain has a mild form of renal disease. None of these newly constructed congenic mutant strains have the severity of proteinuria or the degree of renal pathology characteristic of MRL-lpr mice. Thus, the lpr gene alone is insufficient in producing severe renal injury. The interaction of the lpr gene with other factors is required for the induction of life-threatening lupus nephritis.

Exacerbation of Pneumocystis carinii pneumonia in immunodeficient (scid) mice by concurrent infection with a pneumovirus.

scid mice naturally infected with Pneumocystis carinii and inoculated with a normally apathogenic pneumovirus had significantly higher P. carinii cyst counts and developed significantly more severe P. carinii-related disease than did sham-inoculated, P. carinii-infected scid mice. P. carinii-free, virus-infected scid mice survived for 2 months despite high pulmonary virus titers. These results show that a respiratory virus infection can exacerbate P. carinii disease in an immunocompromised-rodent model.

Four-laser airborne infrared spectrometer for atmospheric trace gas measurements.

We describe the four-laser airborne infrared (FLAIR) instrument, a tunable diode laser absorption spectrometer designed for simultaneous high-sensitivity in situ measurements of four atmospheric trace gases in the troposphere. The FLAIR spectrometer was employed during the large-scale airborne research campaign on tropospheric ozone (TROPOZ II) in 1991 and was used to measure CO, H(2) O(2), HCHO, and NO(2) in the free troposphere where detection limits below 100 parts in 10(12) by volume were achieved.