CT evaluation of caudal versus lumbar access to the intradural space.

BACKGROUND: Percutaneous intraspinal navigation is a new procedure that enables visualization of the central nervous system using a lumbar puncture technique. Past research shows promising potential, but is limited by damage to expensive fiberscopes due to the sharp angle of manipulation at L3-L4. Our objective in this study is to analyse the feasibility of a novel access through the sacral hiatus to eliminate this problem. METHODS: We retrospectively reviewed computed tomography images of the sacrum of 132 subjects, of which nine were excluded either secondary to incomplete imaging of the sacrum or variant anatomy that precluded any possible measurements. Of the remaining 123 patients, we measured kyphotic and lordotic curvature of the sacral canal in comparison to the angle of lumbar puncture. We also measured the anteroposterior diameters of the sacral canal at the distal, middle, and proximal portions. RESULTS: There were no significant differences according to sex or age. The kyphotic angle was a wide angle with a mean value of 167·89±11·71°. The lordotic angle had a mean value of 133·35±7·84°, making it 25·52° more obtuse than the average angle for lumbar puncture. The smallest diameter of the sacral canal was at the sacral hiatus and had a mean value of 4·49±1·66 mm. CONCLUSION: The size and anatomy of the sacral canal is feasible for PIN procedure and appears more favorable as compared to entry via lumbar puncture. The canal opening is wide enough to accommodate most small-diameter fiberscopes without difficulty, and the angles are obtuse enough to limit damage to the expensive fiberscopes.

[The punctum plug as an option for treating retrograde air flow from the lacrimal sac]. / Der Punctum Plug als therapeutische Option des retrograden Aeroflows des Saccus lacrimalis.

BACKGROUND: The causes of keratoconjunctivitis sicca are multifarious. Major causes include eyelid anomalies, Sjögren's disease, injuries, radiation or mucin deficiency. In our case, nasal positive airway pressure due to muscular dystrophy is described as another possible cause. PATIENT: A 32-year-old male patient with advanced Duchenne muscular dystrophy requires nasal continuous positive airway pressure ventilation due to loss of the auxiliary respiratory muscles. The patient presented because permanent air flow from the lower right lacrimal point resulted in epiphora and keratoconjunctivitis sicca on the right side. THERAPY: We reversibly occluded the lower lacrimal duct with a punctum plug flow regulator. The lacrimal flow regulator is available in two sizes, 0.8 and 0.9 mm, and is designed to be inserted into the punctal aperture. The soft silicone plug is delivered with a disposable dilator and can be used on an outpatient basis. It is actually used for punctal occlusion in patients with chronic dry eye syndrome and regulates the flow of lacrimal fluid. After insertion of a 0.9 mm punctal plug, the patient reported marked improvement of the findings, which was confirmed in a follow-up after 4 and 8 weeks. On a visual analog scale of 1 to 10, the subjective status was given a rating of 8. CONCLUSION: By occluding the right lower lacrimal point with a punctal plug, we were able to markedly reduce the unpleasant retrograde air flow and the subjective complaints of the patient with nasal continuous positive airway pressure ventilation.

Organ formation in Drosophila: specification and morphogenesis of the salivary gland.

The Drosophila salivary gland has emerged as an outstanding model system for the process of organ formation. Many of the component steps, from initial regional specification through cell specialization and morphogenesis, are known and many of the genes required for these different processes have been identified. The salivary gland is a relatively simple organ; the entire gland comprises of only two major cell types, which derive from a single contiguous primordium. Salivary cells cease dividing once they are specified, and organ growth is achieved simply by an increase in size of individual cells, thus eliminating concerns about the potential unequal distribution of determinants during mitosis. Drosophila salivary glands form by the same cellular mechanisms as organs in higher organisms, including regulated cell shape changes, cell intercalation and directed cell migration. Thus, learning how these events are coordinated for tissue morphogenesis in an organism for which the genetic and molecular tools are unsurpassed should provide excellent paradigms for dissecting related processes in the more intricate organs of more complicated species.

Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells.

Serum antibody (Ab) can play several roles during B cell immune responses. Among these is to promote the deposition of immune complexes (ICs) on follicular dendritic cells (FDCs). ICs on FDCs are generally thought to be critical for normal germinal center (GC) formation and the development and selection of memory B cells. However, it has been very difficult to test these ideas. To determine directly whether FDC-bound complexes do indeed function in these roles, we have developed a transgenic (Tg) mouse in which all B lymphocytes produce only the membrane-bound form of immunoglobulin M. Immune Tg mice have 10,000-fold less specific Ab than wild-type mice and lack detectable ICs on FDCs. Nonetheless, primary immune responses and the GC reaction in these mice are robust, suggesting that ICs on FDCs do not play critical roles in immune response initiation and GC formation. Moreover, as indicated by the presence and pattern of somatic mutations, memory cell formation and selection appear normal in these IC-deficient GCs.

Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis.

We show here that maintenance of Leishmania infections with Leishmania mexicana complex parasites (Leishmania amazonensis and Leishmania pifanoi) is impaired in the absence of circulating antibody. In these studies, we used mice genetically altered to contain no circulating antibody, with and without functional B cells. This experimental design allowed us to rule out a critical role for B cell antigen presentation in Leishmania pathogenesis. In addition, we show that mice lacking the common gamma chain of Fc receptors (FcgammaRI, FcepsilonRI, and FcgammaRIII) are similarly refractory to infection with these parasites. These observations establish a critical role for antibody in the pathogenesis associated with infection by members of the L. mexicana complex.

A B-cell receptor-specific selection step governs immature to mature B cell differentiation.

Seventy percent of peripheral immature conventional (B2) B cells fail to develop into mature B cells. The nature of this cell loss has not been characterized; the process that governs which immature B cells develop into long-lived peripheral B cells could be either stochastic or selective. Here, we demonstrate that this step is in fact selective, in that the fate of an immature B cell is highly dependent on its Ig receptor specificity. A significant skewing of the B cell receptor repertoire occurs by the time cells enter the mature B cell fraction, which indicates that there is selection of only a minority of immature B cells to become mature B cells. Because only a few heavy-light chain pairs are enhanced of the diverse available repertoire, we favor the idea that selection is positive for these few heavy-light chain pairs rather than negative against nearly all others. Because most immature B cells are lost at this transition, this putative positive selection event is likely to be a major force shaping the mature B cell receptor repertoire available for adaptive immune responses.

A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.

The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51-59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295-1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ-Faslpr (MRL/lpr) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.

Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers.

In the first week of the primary immune response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten, plasmacytic foci and germinal centers (GCs) in C57BL/6 mice are comprised of polyclonal populations of B lymphocytes bearing the lambda1 L-chain (lambda1+). The Ig H-chains of these early populations of B cells are encoded by a variety of VH and D exons undiversified by hypermutation while later, oligoclonal populations are dominated by mutated rearrangements of the VH186.2 and DFL16.1 gene segments. To assess directly Ab affinities within these defined splenic microenvironments, representative VDJ rearrangements were recovered from B cells participating in the early immune response to NP, inserted into Ig H-chain expression cassettes, and transfected into J558L (H-; lambda1+) myeloma cells. These transfectoma Abs expressed a remarkably wide range of measured affinities (Ka = 5 x 10(4)-1.3 x 10(6) M(-1)) for NP. VDJs recovered from both foci and early GCs generated comparable affinities, suggesting that initial differentiation into these compartments occurs stochastically. We conclude that Ag normally activates B cells bearing an unexpectedly wide spectrum of Ab affinities and that this initial, promiscuous clonal activation is followed by affinity-driven competition to determine survival and clonal expansion within GCs and entry into the memory and bone marrow plasmacyte compartments.

A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease.

We have analyzed B cell tolerance in a rheumatoid factor (RF) transgenic mouse model. The model is based on AM14, a hybridoma, originally isolated from an autoimmune MRL/lpr mouse that has an affinity and specificity typical of disease-related RFs from this strain. AM14 binds to immunoglobulin (Ig)G2a of the "a" allotype (IgG2aa) and not to IgG2ab. Thus, by crossing the transgenes onto an IgHa (BALB/c) background or to a congenic IgHb (CB.17) background, we could study the RF-expressing B cells when they were self-specific (IgHa) or when they were not self-specific (IgHb). These features make the AM14 model unique in focusing on a true autoantibody specificity while at the same time allowing comparison of autoreactive and nonautoreactive transgenic B cells, as was possible in model autoantibody systems such as anti-hen egg lysozyme. Studies showed that AM14 RF B cells can make primary immune responses and do not downregulate sIgM, indicating that the presence of self-antigen does not induce anergy of these cells. In fact, IgHa AM14 transgenic mice have higher serum levels of transgene-encoded RF than their IgHb counterparts, suggesting that self-antigen-specific activation occurs even in the normal mouse background. Since AM14 B cells made primary responses, we had the opportunity to test for potential blocks to self-reactive cells entering the memory compartment. We did not find evidence of this, as AM14 B cells made secondary immune responses as well. These data demonstrate that a precursor of a disease-specific autoantibody can be present in the preimmune repertoire and functional even to the point of memory cell development of normal mice. Therefore, immunoregulatory mechanisms that normally prevent autoantibody production must exert their effects later in B cell development or through T cell tolerance. Conversely, the data suggest that it is not necessary to break central tolerance, even in an autoimmune mouse, to generate pathologic, disease-associated autoantibodies.

Contact dermatitis from beryllium in dental alloys.

An increasing number of metals with the potential to cause allergic contact dermatitis have found their way into dental alloys for economic and practical reasons. 2 patients are reported who developed gingivitis adjacent to the Rexillium III alloy in their dental prostheses. Patch testing demonstrated positive reactions to beryllium sulfate, a component of the alloy. Components of dental alloys and the mechanism of the contact dermatitis are discussed.

Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice.

Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.

Structure-function relationships among highly diverse T cells that recognize a determinant from influenza virus hemagglutinin.

We have analyzed the structural and genetic basis for T cell recognition of the complex formed between antigen and class II products of the major histocompatibility complex by performing sequence analysis of T cell receptors (TCRs) induced in response to the helper T cell site 1 of the influenza virus hemagglutinin. The results demonstrate, first, that structurally highly diverse TCRs can be utilized in recognition of the same antigen/I-Ed complex: 12 of 13 TCRs utilize unique V alpha/V beta gene segment combinations, suggesting that approximately 70 different V alpha/V beta combinations are available to BALB/c mice in response to this determinant. Second, comparison of these sequences with the ability of each hybridoma to recognize a panel of peptide analogues suggests that alpha and beta chains of these TCRs frequently determine specificity for the NH2-terminal and the COOH terminal portions, respectively, of the site 1 determinant.

Psoriasis vulgaris: a genetic approach.

Evidence for a genetic contribution in psoriasis comes from direct examination of a large segment of the population in an isolated island environment, epidemiologic and questionnaire studies presented to psoriatic patients, twin studies collected from the literature and from twin registries, and splitsibship analysis. The concordance of psoriasis in monozygotic twins was 65-72%, whereas psoriasis in dizygotic twins was 15-30%. Determination of concordance in older twin pairs from a national twin registry in Denmark revealed nearly 90-100% heritability. In order to link psoriasis with known markers within the human genome, serologic studies have been carried out with a variety of blood group and polymorphic protein antigens. A weak association with the MNS and Lewis Blood Groups Systems (relative risk, 3.5) has been identified. Stronger associations with class I B locus and class II D locus genes (relative risk, 8-12) have also been determined by studies of the human lymphocyte-antigen system. Finally, a strong association with HLA Cw6 has been determined; this marker is thought to be in linkage disequilibrium with B and D locus genes previously associated with psoriasis. The relative risk of developing psoriasis in HLA Cw6 positive individuals is about 24. A few large kindred have been reported in the dermatology literature. These support the hypothesis of autosomal dominant inheritance with penetrance of approximately 60%. In cooperation with The National Psoriasis Foundation, we have now identified over 90 families with psoriasis in three generations. We have begun the process of ascertainment, the construction of family trees, and the collection of leukocyte DNA for linkage analysis with established restriction fragment polymorphisms (RFLP). Our initial assessment is being directed to four RFLP that span approximately 30 centiMorgans of the short arm of human chromosome 6. Although karyotyping is uncommonly done in patients because of psoriasis, we now seek evidence of translocation of chromosome 6 in association with psoriasis.

A large degree of functional diversity exists among helper T cells specific for the same antigenic site of influenza hemagglutinin.

The site-1 determinant of the hemagglutinin molecule of influenza virus (A/PR/8/34) is one of several immunodominant sites in the BALB/c Th cell response to Ha. A synthetic peptide comprising this T cell site (HA110-120), a panel of analogs containing single substitutions in this determinant, and homologs truncated at the amino- or carboxyl-terminal were used to examine the fine specificities of 15 T cells specific for site-1 in the context of I-Ed. The results indicate that every residue within the minimal determinant plays a role in the T cell recognition process, as single substitutions at any of these positions affected the ability of the peptide to stimulate at least some site 1-specific T cells. For the majority of the residues examined, substitutions had dissimilar effects on distinct T cells, indicating that the substituted residues were affecting recognition in a receptor-specific manner. Each of the 15 T cells examined had a distinct fine specificity pattern, suggesting that the BALB/c T cell repertoire for this site is likely to exceed 100 distinct clonotypes.

Kinetic studies and unstructured models of lymphocyte metabolism in fed-batch culture.

The growth of two lymphocyte cell lines, a hybridoma cell line and a human cutaneous T cell lymphoma (HuT78), was studied in fed-batch culture, and unstructured models of growth developed. A criteria was established to insure that the growth rate varied by less than a specified tolerance throughout the culture period. Glutamine and serum were growth-limiting nutrients for both cell lines with half-maximal growth rates at 0. 53 mM glutamine and 0. 55%(v/v) serum for the hybridoma cells and 0. 21 mM glutamine and 1. 5% serum for the HuT-78 cells. Over the range of glucose concentrations from 5. 5 mM to 28 mM, the specific growth rate of hybridoma cells was independent of glucose concentration, whereas glucose concentrations above 5. 5 mM inhibited HuT-78 growth. For both cell lines, the growth rate was significantly inhibited by the addition of ammonium, although the hybridoma cell line was more affected by ammonia than was the HuT-78 cell line. Growth of HuT-78 cells increased in the presence of interleukin-2. Unstructured models for the hybridoma cells were similar to other models presented in the literature. Applications of these models to adoptive immunotherapy are discussed.

Isolation of the MHC genes encoding the tumour-specific class I antigens expressed on a murine fibrosarcoma.

The C3H UV-induced fibrosarcoma, 1591, is highly immunogenic and, therefore, is readily rejected when transplanted into immunocompetent syngeneic recipients. Previous analysis of 1591 with tumour-specific or H-2-reactive monoclonal antibodies revealed that this antigenicity might be due to the expression of two novel class I major histocompatibility complex (MHC) antigens. In this report we describe the molecular cloning and initial characterization of three genes which account for all of the unique serological class I reactivities observed on this tumour. These include two distinct, but highly conserved, H-2L-like genes, and a third gene the product of which bears determinants which are characteristic of both the tumour and of class I products of the H-2k haplotype. Moreover, each of these genes contains a polymorphic restriction enzyme fragment which is detected in the class I sequences of 1591 relative to normal C3H tissue. Since the expression of these polymorphic class I sequences is relevant to the immunogenicity of 1591, the mutational events by which these genes were generated may be significant to the immunobiology of this tumour.

Retroviral-associated eosinophilic leukemia in the cat.

Fourteen specific-pathogen-free cats were inoculated with a putative env gene recombinant feline retrovirus, PR8. An isolate of the Rickard strain of feline leukemia virus (FeLV-R), PR8, has the properties of both an exogenous (FeLV-R) and an endogenous (xenotropic) feline retrovirus (RD-114). Twelve of the PR8-inoculated cats developed viremia; 2 of the 12 cats developed eosinophilia, with 1 being diagnosed with eosinophilic leukemia and the other with extreme eosinophilic hyperplasia. Eosinophilic leukemia is rare in cats and has not previously been associated with retroviral infection. Changes in the viral envelope properties may have altered the pathogenicity of the exogenous virus to cause this rare form of leukemia.