Junctional Region Research Articles

Localization of the Thyroid Peroxidase Autoantibody Immunodominant Region to a Junctional Region Containing Portions of the Domains Homologous to Complement Control Protein and Myeloperoxidase

Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP- and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP- and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO.

The in vitro generation of Ph1+ ALL-specific HLA-A24-restricted cytotoxic T lymphocytes using a synthetic 16 mer minor bcr–abl peptide

Sixteen mer peptide, which spans the junctional region of the acute lymphoid leukemia (ALL)-specific minor bcr–abl fusion protein and contains a motif that can bind to human leukocyte antigen (HLA)-A24, was constructed. We tried to generate Philadelphia chromosome 1 (Ph1) positive ALL-specific cytotoxic T lymphocytes (CTLs) from eight normal HLA-A24+ individuals with peptide-pulsed autologous dendritic cells (DCs). CTLs could be generated from the mononuclear cells (MNCs) of a single donor, which could kill peptide-pulsed autologous DCs and two A24+ ALL lines, while an HLA-A24+ CML line was only weakly killed and unpulsed DCs or the control lines Daudi or K562 were not recognized. Those CTLs consisted predominantly of CD8+ T cells whose cytotoxicity could be neutralized by monoclonal antibodies to HLA-class I or HLA-A24, and also produced interferon (IFN)-γ after being stimulated with peptide-pulsed DCs.

Crumbs interacts with moesin and beta(Heavy)-spectrin in the apical membrane skeleton of Drosophila.

The apical transmembrane protein Crumbs is necessary for both cell polarization and the assembly of the zonula adherens (ZA) in Drosophila epithelia. The apical spectrin-based membrane skeleton (SBMS) is a protein network that is essential for epithelial morphogenesis and ZA integrity, and exhibits close colocalization with Crumbs and the ZA in fly epithelia. These observations suggest that Crumbs may stabilize the ZA by recruiting the SBMS to the junctional region. Consistent with this hypothesis, we report that Crumbs is necessary for the organization of the apical SBMS in embryos and Schneider 2 cells, whereas the localization of Crumbs is not affected in karst mutants that eliminate the apical SBMS. Our data indicate that it is specifically the 4.1 protein/ezrin/radixin/moesin (FERM) domain binding consensus, and in particular, an arginine at position 7 in the cytoplasmic tail of Crumbs that is essential to efficiently recruit both the apical SBMS and the FERM domain protein, DMoesin. Crumbs, Discs lost, βHeavy-spectrin, and DMoesin are all coimmunoprecipitated from embryos, confirming the existence of a multimolecular complex. We propose that Crumbs stabilizes the apical SBMS via DMoesin and actin, leading to reinforcement of the ZA and effectively coupling epithelial morphogenesis and cell polarity.

Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom

The occurrence of extended tight junction (TJ) structures, including zonulae occludentes (ZO), and the spatial arrangement of TJ proteins in stratified mammalian epithelia has long been controversially discussed. Therefore, we have systematically examined the localization of TJ proteins in diverse stratified epithelial tissues (e.g., epidermis, heel pad, snout, gingiva, tongue, esophagus, exocervix, vagina, urothelium, cornea) of various species (human, bovine, rodents) as well as in human cell culture lines derived from stratified epithelia, by electron microscopy as well as by immunocytochemistry at both the light and the electron microscopic level, using antibodies to TJ proteins such as occludin, claudins 1 and 4, protein ZO-1, cingulin and symplekin. We have found an unexpected diversity of TJ-related structures of which only those showing colocalization with the most restricted transmembrane TJ marker protein, occludin, are presented here. While in epidermis and urothelium occludin is restricted to the uppermost living cell layer, TJ-related junctions are abundant in the upper third or even in the majority of the suprabasal cell layers in other stratified epithelia. Interfollicular epidermis contains, in the stratum granulosum, extended, probably continuous ZO-like structures which can also be traced at least through the Henle cell layer of hair follicles. Similar apical ZO-like structures have been seen in the upper living cell layers of all other stratified epithelia and cell cultures examined, but in most of them we have noticed, in addition, junctional regions showing relatively broad, ribbon-like membrane contacts which in cross-section often appear pentalaminar, with an electron-dense middle lamella ("lamellated TJs", coniunctiones laminosae). In suprabasal layers of several stratified epithelia we have further observed TJ protein-containing junctions of variable sizes which are characterized by a 10-30-nm dense lamina interposed between the two membranes ("sandwich junctions"; iuncturae structae). Moreover, we have often observed variously sized regions in which the intermembrane distance is rather regularly bridged by short rod-like elements ("cross-bridged cell walls"; parietes transtillati), often in close vicinity of TJ-related structures or desmosomes. The significance of these structures and their possible biological importance are discussed.

Over-expression of PAR-3 suppresses contact-mediated inhibition of cell migration in MDCK cells.

PAR-3 is one of the PAR proteins, previously named ASIP, which are indispensable for the establishment of cell polarity in the embryo as well as differentiated epithelial cells. In mammalian epithelial cells, it forms a ternary complex with aPKC and PAR-6, and is localized to the tight junction that has been suggested as being important for creating cell polarity. To gain insights into the mode of PAR-3 function in mammalian epithelial cells, we examined the effect of PAR-3 over-expression in MDCK cells. Although exogenous PAR-3-expression does not affect the epithelial polarity of confluent cells, it drastically transforms the morphology of cells at low density into a fibroblastic form with developed membrane protrusions. Time-lapse observations have revealed that PAR-3 over-expressing cells show intense motility, even after they have assembled into loose colonies, suggesting that the contact-mediated inhibition of cell migration (CIM) is suppressed. The expressions of E-cadherin and vimentin do not change with PAR-3 over-expression, suggesting that exogenous PAR-3 only disturbs the endogenous equilibrium of cellular states between a fundamental fibroblastic structure and an epithelial one. The co-expression of a dominant negative mutant of Rac1 and the addition of nocodazole strongly antagonize the effect of PAR-3 over-expression, suggesting the involvement of Rac1 activation and microtubule polymerizations. : The data presented here suggest an intriguing link between the contact-mediated inhibition of cell migration and the regulation of cell polarity. The putative PAR-3 activities demonstrated here may function endogenously in the epithelial cell polarization process by being sequestered from the cytosol to the cell-cell junctional regions with aPKC and PAR-6 upon cell-cell adhesion.

Influence of age on cervicothoracic spinal curvature: An ex vivo radiographic survey

Objective. To define the post-mortem cervicothoracic spinal curvature relative to age. Design. Spinal curvature assessment of lateral cervicothoracic radiographs. Background. A late consequence of age is the progressive accentuation of spinal curvatures, particularly the thoracic kyphosis. Little is known about the influence of the kyphosis on the alignment of the cervical spine. Method. One hundred and seventy two lateral spinal radiographs (113 males, 59 females) were analysed using two procedures: (1) sagittal curve deformation angles were derived, according to the method of Cobb, for thoracic (T1–T12), cervical (C2–C7) and cervicothoracic junctional regions (C6–T4); and (2) the cervicothoracic curvatures were digitised (C2–T12), to derive the apex of both curves and the inflexion point. Results. A significantly increasing thoracic spinal curvature was determined for both genders, with the mean apex of the kyphosis close to T6. The cervical lordosis tended to flatten with increasing age, particularly in males, with the cervical apex location shifting cranially. This association was significant in older males and females. The mean location of the cervicothoracic curve inflexion point moved from T3 towards C7–T1 with increasing age. Conclusion. The cervicothoracic spinal curvature undergoes progressive change through the lifespan with a subsequent cranial migration of the inflexion point between the thoracic kyphosis and cervical lordosis, accompanied by a similar shift in the cervical apex. Relevance Sensitive measures of spinal curvature have utility in determining changes attributed to age, deformity or trauma on cervicothoracic spinal alignment. The value of assessing the location of curve inflexion lies in the ability to quantify changes in the relationship between different regions of the human spine without problems associated with identifying specific vertebral landmarks.

Comparison of the effect of pulsatile and nonpulsatile shear stress on adherens junctions in human endothelial cells

In vivo, endothelial cells experience shear stress resulting from pulsatile flow. Many publications have shown that the pulsatile nature of this flow influences endothelial function and structure including intracellular calcium concentrations, cell shape and actin stress fibre formation. A recent in vitro study using bovine aortic cells showed exposure to nonpulsatile flow caused transient disruption of adherens junctions through perturbation of VE‐cadherin and the disassembly of the actin cytoskeleton. The aim of this study was to observe the responses of the adherens junctional molecule VE‐cadherin, and F‐actin, to pulsatile and nonpulsatile flow in human endothelial cells over a 48‐h period.Human umbilical vein endothelial cells (HUVEC) were isolated from fresh umbilical cords taken from elective caesarean sections (n = 6). At passages 2–4 the cells were seeded onto glass coverslips pretreated with 3‐aminoproryaltriethoxysilane (APES). 48 h after reaching confluence the cells were transferred to a parallel plate flow chamber and exposed to slowly increased pulsatile or nonpulsatile laminar flow, producing a final shear stress of 1.5 dynes/cm2. Cells were left under flow for up to 48 h. At t = 0, 8 and 48 h immunocytochemistry was used to visualise VE‐cadherin and phalloidin‐TRITC to visualise F‐actin.At t = 0, VE‐cadherin was localised at cell‐cell contacts and the cells contained randomly oriented stress fibres and cortically located perijunctional actin. Following 8 h exposure to both types of flow no VE‐cadherin or actin could be detected. After 48 h exposure VE‐cadherin and F‐actin had returned to the junctional regions of the cells exposed to nonpulsatile flow. By comparison in cells exposed to pulsatile flow VE‐cadherin was absent from the cell periphery but was found diffusely throughout the cell. Actin, both stress fibre and cortical, was absent from these cells.These results show that reassembly of the adherens junction, with a return of VE‐cadherin to cell‐cell contacts and a reformation of the actin cytoskeleton, is only seen in cells that have been exposed to nonpulsatile flow. Further work will show whether such adaptations are merely delayed under pulsatile flow or are prevented from occurring entirely. This study highlights the importance of using pulsatile flow when studying endothelial behaviour.The financial support of EPSRC and Sulzer Vascutek Ltd is gratefully acknowledged.

Genetic analysis of the rgg-gtfG junctional region and its role in Streptococcus gordonii glucosyltransferase activity.

Glucans synthesized by glucosyltransferase enzymes of oral streptococci facilitate bacterial accumulation on surfaces. The Streptococcus gordonii glucosyltransferase gene, gtfG, is positively regulated by rgg, which encodes a putative cytoplasmic protein. The gtfG promoter and ribosomal binding sequences are located within a DNA inverted repeat immediately downstream of rgg. Polycistronic rgg-gtfG as well as rgg- and gtfG-specific transcripts are associated with this chromosomal region. Previous studies have shown that the rgg product acts in trans near the gtfG promoter to increase the level of gtfG transcript, but it does not affect the level of rgg-gtfG transcript. To further analyze regulation by rgg, a series of strain Challis derivatives was constructed and glucosyltransferase activities were determined. Strains in which rgg was separated from gtfG by integrated vector sequences had decreased levels of glucosyltransferase activity; plasmid-borne rgg could not increase activity to parental levels. As expected, strains with chromosomal deletions involving the rgg structural gene and either the rgg or gtfG promoter also showed decreased glucosyltransferase activity. Plasmid-borne rgg could increase glucosyltransferase activity only in strains which had a 36-bp chromosomal region beginning 72 nucleotides upstream of the gtfG transcriptional start site. Results suggest that these nucleotides, located within the 3' end of rgg, are necessary, either by direct involvement in binding or by indirectly affecting secondary structure, for Rgg to increase glucosyltransferase activity. Surprisingly, the presence of the rgg promoter upstream of this 36-bp region significantly increased the effects of plasmid-borne rgg. Implications for glucosyltransferase regulation and applicability to other rgg-like determinants are considered.

Minimal residual disease in Brazilian children with acute lymphoid leukemia: comparison of three detection methods by PCR

The minimal residual disease (MRD) detection by the polymerase chain reaction (PCR) in children with acute lymphoblastic leukemia has been pointed to be an adverse prognostic factor. Detection methods based on this technique using clone-specific primers are cumbersome and time consuming. The detection of monoclonal gene rearrangements of gamma T-cell receptors (TCRγ) is a simpler although less sensitive method. In the present study, we analyzed the presence of MRD during four different phases of treatment (week 4; 3–6, 12–24 months, and end of treatment) in 34 Brazilian children with lymphoid leukemia by three detection methods based on the PCR technique: (1) using consensus primers for the detection of a clonal population for TCRγ; (2) clone-specific primers for the junctional region of TCRγ; and (3) a semi-nested reaction with an initial cycle with consensus primers followed by a second cycle with clone-specific primers. MRD presence was associated with a shorter event-free survival and was the major independent prognostic factor in most of the phases analyzed. The use of consensus primers for the detection of TCRγ clonality, although less sensitive, proved to be a simpler, faster and less costly method whose positivity was associated with more than 90% relapse rates during all phases analyzed.

Concomitant lower thoracic spine disc disease in lumbar spine MR imaging studies.

Our objective was to study the coexistence of lower thoracic-spine disc changes in patients with low back pain using a large field of view (FOV) in lumbar spine MR imaging. One hundred fifty patients with low back pain were referred to an MR examination. All patients were studied with a large FOV (27 cm), covering from the coccyx to at least the body of T11. Discs were coded as normal, protrusion, and extrusion (either epiphyseal or intervertebral). The relationship between disc disease and level was established with the Pearson chi(2) test. The T11-12 was the most commonly affected level of the lower thoracic spine with 58 disc cases rated as abnormal. Abnormalities of T11-12 and T12-L1 discs were significantly related only to L1-L2 disease ( p=0.001 and p=0.004, respectively) but unrelated to other disc disease, patient's gender, and age. No correlation was found between other discs. Magnetic resonance imaging of the lumbar spine can detect a great amount of lower thoracic disease, although its clinical significance remains unknown. A statistically significant relation was found within the thoracolumbar junctional region (T11-L2), reflecting common pathoanatomical changes. The absence of relation with lower lumbar spine discs is probably due to differences in their pathomechanisms.

Clinical pharmacology: drugs as a benefit and/or risk in sudden unexpected death in epilepsy?

Death may be the consequence of natural or unnatural causes, such as accidents, homicide, and suicide, which have no relationship to the disease of epilepsy. Direct causes of death include status epilepticus, and indirect causes may be head trauma or drowning subsequent to a seizure. When death occurs suddenly and without explanation, the term sudden unexpected unexplained death is used. Unexplained is a term that clinicians and research scientists are working to clarify. Numerous preclinical animal studies have been conducted as models for sudden death and have led to clinical studies in persons with epilepsy. These studies show that sympathetic nerve stimulation, ouabain, or coronary occlusion increased temporal dispersion of recovery of ventricular excitability and led to an underlying electrical instability that predisposed the ventricularmyocardium to arrhythmia. Cardiac arrhythmias in an animal model for ouabain-induced toxicity were associated with neural autonomic dysfunction. Neural discharges were characterized by increases, decreases, or no change in the discharge of postganglionic cardiac sympathetic nerves monitored simultaneously, predisposing to cardiac arrhythmia. Stimulation of the sympathetic ventrolateral cardiac nerve produced a shift in the origin of the pacemaker and tachyarrhythmias because the nerve is not uniformly distributed to the various regions of the heart but is localized to the atrioventricular junctional and ventricular regions. Such nonuniform distribution of sympathetic nerves would also contribute to initiation of arrhythmia as a nonuniform neural discharge occurred. Studies examining the physiology and pharmacology of this finding in multiple animal models found that subconvulsant, interictal discharge was associated with autonomic cardiac neural non-uniform discharge and cardiac arrhythmias. As a result of further investigations, Lathers and Schraeder edited a book in 1990 that summarized the clinical problem of sudden unexpected death and epilepsy (SUDEP). The contributors concluded that there was a paucity of clinical data addressing the mechanism of death. Regulatory response resulting from the consequent increased awareness of SUDEP occurred in 1993, when the FDA focused attention of practitioners and pharmaceutical manufacturers on the question of whether use of anticonvulsant drugs contributes to or prevents sudden unexpected death in epileptic persons. The FDA-convened panel of scientists considered the prevalence of sudden unexpected death in patients involved in studies associated with developing new anticonvulsant drugs and reviewed data on the risk of sudden unexpected death in patients taking lamotrigine. The risk of SUDEP was no different from thatfound in the young epilepsy population in general. Estimated SUDEP rates in patients receiving the new anticonvulsant drugs lamotrigine, gabapentin, topiramate, tigabine, and zonisamide were found to be similar to those in patients receiving standard anticonvulsant drugs, suggesting that SUDEP rates reflect population rates and not a specific drug effect. The FDA required warning labels on the risk of SUDEP in association with the use of each of the above-mentioned drugs. Another effect of bringing SUDEP to the attention of epilepsy researchers has been the expansion of basic science and the development of epidemiological and clinical studies directed at this phenomenon. Results from some of these studies are discussed in this article.

Detection of Barrett’s epithelium by acoustic microscopy

Barrett’s esophagus is associated with increased risk of adenocarcinoma of the gastroesophageal junctional region. The presence of goblet cells (intestinal metaplasia) in columnar cell-lined esophageal mucosa defines Barrett’s change. The diagnosis of Barrett’s esophagus is based on the presence of intestinal metaplasia in a biopsy from an endoscopically visualized abnormal columnar epithelium. In this pilot study, acoustic microscopy was used to identify the mucosal structure of 10 distal esophageal biopsies. Sections cut at 5 μm of archival paraffin blocks on glass slides were used for this study. Acoustic microscopy permitted the identification of low- and high-power images of epithelial architecture and cellular detail, including Barrett’s epithelium. This modality of visualization has the potential to detect lesions such as Barrett’s metaplasia, low- and high-grade dysplasia and early carcinoma. If it can be applied to in vivo endoscopy, acoustic microscopy has the potential to increase the accuracy of the diagnosis of Barrett’s esophagus, dysplasia and malignancy by providing a method of accurately directing biopsies at endoscopy.

Oligoclonally expanding gammadelta T lymphocytes induce IgA switching in IgA nephropathy.

The aetiology of IgA nephropathy (IgAN) is closely related with abnormality of mucosal immunity. We investigated the roles of gammadelta T cells in the regulation of IgA production by B cells in IgAN patients. The proportion of gammadelta T cells in peripheral blood mononuclear cells (PBMNC) was higher in IgAN patients than in the controls and was found to be correlated with the proportion of surface IgA-positive (sIgA+) B cells, which are precursors of IgA-secreting plasma cells. After in vitro PWM stimulation, sIgA expression on B cells and IgA production were significantly enhanced in PBMNC obtained from IgAN patients, whereas the enhancements were abolished by removal of gammadelta T cells from the PBMNC. Purified gammadelta T cells from IgAN patients induced surface IgA expression on naïve sIgD+ B cells more effectively than did alphabeta T cells. Moreover, stimulated gammadelta T cells from IgAN patients produced a larger amount of TGF-beta1, which is one of the main cytokines that induces IgA class switching on B cells, as compared with alphabeta T cells and control gammadelta T cells. The expanded gammadelta T cells from IgAN patients exclusively expressed Vgamma9, and the nucleotide sequences of junctional regions of Vgamma9 showed very limited TCR diversities. It was therefore concluded that gammadelta T cells, which are expanded in response to specific antigens, enhance IgA class switching on B cells in IgAN patients.

Microvascularization of the mucocutaneous junction of the eyelid in fetuses and neonates.

The aim of this study was to specify the microvascularization of the junctional region between the integuments of the superficial surface of the free margin of the eyelid and the palpebral conjunctival mucosa. The study was carried out using histological or transparified slices of upper and lower eyelids taken from fetuses and neonates, in which the vascular system was injected with agarized China ink. The mucocutaneous junction of the eyelid is located at the posterior border of the free margin and extends to its deep surface. It has vascular similarities to the oral cavity and the nasal pyramid. Under a thickened avascular epithelium, there is a papillary network composed of characteristic loops that are less raised than in the lips but more developed than in the nose. The superficial and deep vascular reticular networks are comparable in fundamental arrangement to those of other junctional zones. Thus, the palpebral mucocutaneous junction shows cutaneous-type microvascularization, just like the other junctional zones of the head.

Abnormal formation of sarcoplasmic reticulum networks and triads during early development of skeletal muscle cells in mitsugumin29-deficient mice.

Recently, we detected a novel membrane protein, mitsugumin29 (MG29), in the triads in rabbit skeletal muscle cells and suggested important roles for this membrane protein in the formation of the sarcoplasmic reticulum (SR) networks and triads in muscle cells. In the present study, we examined the development of skeletal muscle cells in MG29-deficient mice to try to determine the roles played by MG29 in the formation of the SR networks and triads. Ultrastructural observations revealed some morphological abnormalities in these mice, such as incomplete formation of the SR networks, an irregular running of the transverse tubule and a partial defect in the triads at the A-I junctional region. These ultrastructural abnormalities occurred during early myogenesis and were preserved until the adult stage. The possible roles for MG29 in the formation of SR networks and triads in skeletal muscle cells are discussed in the light of these observations.

Molecular Evolution of Catalytic Antibodies in Autoimmune Mice

Catalytic Abs (catAbs) preferentially evolved in autoimmune MRL/MPJ-lpr/lpr (MRL/lpr) mice upon immunization with the phosphonate transition-state analogue (TSA), but this did not happen in normal BALB/c mice. The majority of the catAbs from MRL/lpr mice were from several independent clones of the same family. Most of them had a lysine at position 95 in the heavy chain (H95), which is at the junctional region. This residue, which interacts with the phosphonate moiety of the TSA and presumably is involved in the catalytic activity, was not changed even after expansive evolution following multiple mutations. By contrast, the majority that arose from BALB/c mice were the non-catAbs, which were quite different in the sequence from the catAbs from MRL/lpr mice, but they were clonally related to one another, so most of them were originated from a single clone. In the MRL/lpr mice, the catalytic subsets that existed in the initial repertoire were effectively captured by the phosphonyl oxygens in the TSA by interacting with the lysine at H95. In the BALB/c mice, however, another noncatalytic subset with only the binding capability directed to a moiety other than the phosphonate moiety was alternatively evolved, because of the lowest abundance or elimination of the catalytic subsets.

Dominant Lesional T Cell Receptor Rearrangements Persist in Relapsing Psoriasis but are Absent from Nonlesional Skin: Evidence for a Stable Antigen-Specific Pathogenic T Cell Response in Psoriasis Vulgaris

In a previous study we reported that clonally expanded T cell receptor beta-chain rearrangements characterized the T cell receptor usage in skin lesions of psoriasis vulgaris and indicated antigen-specific T cell selection. To assess the relevance of clonal T cell expansion for disease progression, we now determined if select clonal T cell receptor rearrangements persisted over time and were present in nonlesional skin. Sequential biopsies were taken from psoriatic skin lesions of two patients. V-D-J junctional regions of T cell receptor beta-chain variable region gene families 2, 3, 6, 13S1, and BV17 were cloned and sequenced, as these particular BV gene families are preferentially selected in psoriatic skin lesions. The lesional T cell receptor rearrangements were compared with the T cell receptor usage in nonlesional skin and in blood. Several T cell receptor beta-chain rearrangements with high transcript frequency in the first lesional biopsy were again found in sequential lesional biopsies taken as much as 3 y later from psoriasis relapses. Only T cell receptor beta-chain rearrangements with low transcript abundance showed variability in that several clones appeared for the first time or disappeared. Although nonlesional skin also exhibited a restricted T cell receptor usage with clonal T cell receptor rearrangements, the T cell receptor usage in lesional and nonlesional skin differed nearly completely. The select lesional recurrence of identical T cell receptor rearrangements reveals that inflammation in psoriasis involves the same clonally expanded T cell populations and the same antigens over prolonged periods of time. It hereby suggests that specifically recruited and locally expanded T cell clones are permanently involved in psoriatic inflammation and may play a crucial part in disease perpetuation.

Infiltration of canonical Vgamma4/Vdelta1 gammadelta T cells in an adriamycin-induced progressive renal failure model.

We have previously reported an infiltration of renal interstitial gammadelta T cells in Adriamycin-induced progressive glomerulosclerosis in the rat kidney. The TCR repertoire and sequences used by these gammadelta T cells have now been studied. Two injections of Adriamycin 14 days apart caused segmental glomerulosclerosis, massive interstitial infiltration of mononuclear cells, and end-stage renal failure. Flow cytometry of lymphocyte subpopulations with Abs to CD3, the gammadelta TCR, and the alphabeta TCR showed that gammadelta T cells as a proportion of CD3(+) cells were increased in Adriamycin-treated kidneys (8.5 +/- 5.4%), but not in lymph nodes (1.3 +/- 0.4%). A semiquantitative score of glomerular damage (r = 0.65; p < 0.01) and creatinine (r = 0.62; p < 0.01) correlated significantly with the presence of gammadelta T cells. TCR Vgamma repertoire analysis by RT-PCR and Southern blotting showed that Vgamma2 was the dominant subfamily in lymph nodes, whereas Vgamma4 became the predominant subfamily in advanced stages of the rat Adriamycin-treated kidney. Sequencing of the Vgamma4-Jgamma junctional region showed an invariant sequence. The amino acid sequence of the junctional region of the Vgamma4 TCR was the same as the reported mouse canonical Vgamma4 TCR sequence. Analysis of the kidney Vdelta repertoire showed dominant expression of Vdelta1, and sequencing again revealed the selective expression of a canonical Vdelta1 gene. Semiquantitative RT-PCR for cytokine gene expression showed that gammadelta T cells from the kidneys expressed TGF-beta, but not IL-4, IL-10, or IFN-gamma. These results suggest that the predominant gammadelta T cells in the Adriamycin kidney use an invariant Vgamma4/Vdelta1 receptor.

Emergence of a type II collagen-specific helper T cell response.

Antigen-driven development of persistent self-reactive Th cells underlies the chronic, progressive nature of many autoimmune diseases. It is crucial to understand the behavior of these self-reactive Th cells; however, they have been notoriously difficult to isolate directly ex vivo. Collagen-induced arthritis (CIA) can be initiated in I-A(q)-expressing mice (DBA/1) using heterologous type II collagen (cII) immunization and is dependent on Th cells that are specific for a single immunodominant epitope. Here, we identify one compartment of cII-specific Th cell using TCR beta expression, cell surface phenotype, and direct single-cell repertoire analysis. A subpopulation of CD4(+)V beta 10(+) T cells up-regulates both CD44 and GL7 and expands significantly in response to initial priming in the majority of animals (D9: 70%). The cII-specific V beta 10(+) primary responders are further resolved through expression of a highly restricted junctional region, previously associated with autoimmune disease. This cII-specific clonotype rapidly re-expands upon antigen recall and can be isolated from the lymph nodes of arthritic animals. These single-cell analyses quantify the emergence, decline and rapid re-emergence of a self-reactive Th cell population in vivo and outline one strategy for isolating these cells directly ex vivo.

Electron-microscopic and immunohistochemical studies of eyelid reopening in the mouse.

The development of the eyelids was studied in the mouse from birth to day 12. In transmission electron microscopy (TEM), the desmosome-mediated cell-to-cell adhesions were clearly observed from day 0 to 10 after birth in the junctional region where the upper and lower eyelids fuse. The keratinizing cells in the junctional region, especially in the middle area where the separation occurs by about day 12, gradually elongated toward both surfaces of the fused eyelid with development. In immunohistochemistry, the existing pattern of keratin-positive cells showed distinct developmental changes. Expression of keratin 1 started to extend from the epidermal surface to the junctional region by day 4, and reached the conjunctival part of the junctional region by about day 8. After day 10 keratin 1 began to disappear from the epidermal surface, and by about day 12 keratin 1 almost disappeared from the entire epithelium of the eyelids. Expression pattern of keratin 7 was very similar to that of keratin 1. These findings indicate that epithelial cell differentiation progresses from the skin through to the junctional region to conjunctiva, which may play a role in the eyelids, separation.