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Merging developments in RFID and wireless sensor networks have increased the interest in using backscatter radio for tag-to-tag communication. Low power consumption is a major concern, often limiting the choice to incoherent receivers such as diode envelope detectors. Link budget calculations require receiver sensitivity specifications, however accurate characterizations are scarce. In this work, we show that the receiver sensitivity is strongly related to the exciter incident power. Full-range analysis of a zero-bias diode detector shows a decreasing noise factor at higher incident powers, which leads to improved sensitivity. On the other side, at strong incident powers, the phase noise of the exciter interferes with the received signal, setting an lower bound to the sensitivity of the backscatter receiver. This work proposes a semi-passive RFID tag capable of 1kbps to 10kbps data rates in the 434MHz band using off the shelf components. The tag has a low power consumption of 85μW during receive, 45μW during transmit and less than 4μW in sleep. The receiver sensitivity at 1kbps shows a 25dB variation as a function of exciter incident power between -50 to 10dBm, while a peak sensitivity of around -80dBm is measured at an incident power of -25dBm.

Three new ‘ Aphyosemion ’ species are described from the upper Louesse River in the Massif du Chaillu, Republic of the Congo, based on a combination of DNA, habitat preference, male colour pattern, and morphological data. ‘ Aphyosemion ’ cyanoflavum , new species, is a member of the ‘ A ’ . ogoense group. It differs from its congeners by a unique colour pattern and cephalic sensory system which contains a wide supra-orbital groove with large, densely pigmented anterior neuromasts and dark frontal neuromasts housed in one pit with one central anterior lobe. ‘ Aphyosemion ’ mandoroense , new species, and ‘ A ’ . cryptum , new species, are members of the ‘ A ’ . coeleste group. ‘ Aphyosemion ’ cryptum , new species, is in appearance very similar to ‘ A ’ . coeleste , but lacks the typical post opercular metallic green blotch and is generally larger in body size. Initial DNA analyses demonstrate that ‘ A ’ . cryptum, new species , is more closely related to ‘ A ’ . mandoroense , new species, than to ‘ A ’ . coeleste, despite similarity in appearance . ‘ Aphyosemion ’ cryptum, new species, and ‘ A ’ . coeleste occur syntopic in several locations in a sub-catchment of the upper Louesse system, however differ in microhabitat preference. ‘ Aphyosemion ’ mandoroense, new species, differs by male body and fin colour pattern from all species of the ‘ A ’ . coeleste group except ‘ A ’ . citrineipinnis . From the latter, it can be distinguished by the absence of red pigmentation and a dark grey to black margin in the unpaired fins.

A new procatopodid, assigned to the genus Poropanchax, is described from a wetland habitat located adjacent to the Inga Falls in Lower Congo. Poropanchax pepo, new species, is distinguished from all congeners by a combination of characters including a higher D/A ratio, rounded anal and dorsal fins, a humeral blotch in males, and the absence of a sharp ventral process on the basipterygium. The finding of a Poropanchax species in Lower Congo was unexpected, since the closest known congeneric population is located in northwestern Gabon, some 700 kilometers to the north. As recognized herein the genus Poropanchax is a clade comprised of five species geographically restricted to humid coastal regions of west and west-central Africa.

Co-existence problems in a mobile terminal environment pose strict requirements on the linearity of a front-end receiver. In this paper, active feedback is explored as a means to relax such requirements by providing channel selectivity as early as possible in the receiver chain. The proposed receiver architecture addresses some of the most common problems of integrated RF filters, while maintaining their inherent tunability. Through a simplified and intuitive analysis, the operation of the receiver is examined and the design parameters affecting the filter characteristics, such as bandwidth and stop-band rejection, are determined. A systematic procedure for analyzing the linearity of the receiver reveals the possibility of LNA distortion canceling, which decouples the trade-off between noise, linearity and harmonic radiation. A prototype designed in a standard 65nm CMOS process occupies < 0:06mm2 and utilizes an RF channel-select filter with a 1-to-2:5GHz tunable center frequency to achieve 48dB of stop-band rejection and a wideband IIP3 > +12dBm.

Objective— HSP60-specific T cells contribute to the development of the immune responses in atherosclerosis. This can be dampened by regulatory T cells activated via oral tolerance induction, and we explored the effect of oral tolerance induction to HSP60 and the peptide HSP60 (253 to 268) on atherosclerosis. Methods and Results— HSP60 and HSP60 (253 to 268) were administered orally to LDLr −/− mice before induction of atherosclerosis and resulted in a significant 80% reduction in plaque size in the carotid arteries and in a 27% reduction in plaque size at the aortic root. Reduction in plaque size correlated with an increase in CD4 + CD25 + Foxp3 + regulatory T cells in several organs and in an increased expression of Foxp3, CD25, and CTLA-4 in atherosclerotic lesions of HSP60-treated mice. The production of interleukin (IL)-10 and transforming growth factor (TGF)-β by lymph node cells in response to HSP60 was observed after tolerance induction. Conclusion— Oral tolerance induction to HSP60 and a small HSP60-peptide leads to an increase in the number of CD4 + CD25 + Foxp3 + regulatory T cells, resulting in a decrease in plaque size as a consequence of increased production of IL-10 and TGF-β. We conclude that these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) may provide new therapeutic approaches for the treatment of atherosclerosis.

Immunosterilization is an attractive alternative to surgical castration. Gonadotropin-releasing hormone (GnRH) controls the production of the gonadotropins thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis. Induction of neutralizing antibody can abrogate the effect of the hormone. Current GnRH-based vaccines often require strong adjuvants and/or multiple injections of the vaccines to overcome variability in the response. Heat shock proteins (hsp) have been used as carrier molecules because of their powerful intrinsic ability to enhance an immune response to associated antigens. A GnRH-analogue, GnRH-d6-Lys, was conjugated to recombinantMycobacterium tuberculosishsp70. Male BALB/c mice were immunized i.p. with GnRH-hsp70 in the mild adjuvant Ribi or in incomplete Freund’s adjuvant (IFA). The initial immunizations were done on pre-pubertal 3-week-old mice, with boosts at 5 and 8 weeks of age. The mice were killed at 10 weeks of age and GnRH-specific antibodies and serum testosterone levels measured. All the immunized mice mounted GnRH-specific antibody responses, with no difference in the mice immunized with GnRH-hsp70/Ribi or with GnRH-hsp70/IFA. There was substantial atrophy of the urogenital complex and significantly (P< 0.0005) reduced levels of testosterone-dependent testicular relaxin-like factor mRNA expression. Mice immunized with GnRH-hsp70/Ribi showed substantially reduced (P< 0.001) serum testosterone levels. These results indicate that hsp70 may serve as a particularly advantageous carrier for GnRH-based vaccines.

Objective: To obtain more insight in the role of IGF-1 in cardiac remodeling and function after experimental myocardial infarction. We hypothesized that cardiac remodeling is altered in IGF-1 deficient mice, which may affect cardiac function. Methods: A myocardial infarction was induced by surgical coronary artery ligation in heterozygous IGF-1 deficient mice. One week after surgery, left ventricular function was analyzed, and parameters of cardiac remodeling were measured. Results: No significant difference in cardiac function was found between infarcted wildtype and knock-out animals, despite a marked reduction in capillarization and blunting of the hypertrophic response of the interventricular septum in the IGF-1 deficient group. Furthermore, decreased DNA synthesis and increased apoptosis rates were observed in the IGF-1 knock-out mice. Conclusion: IGF-1 deficient mice show preservation of cardiac function 1 week after MI, despite an altered cardiac remodeling process.

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4+/−- and HLA-DR1+/+-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Background —Nebivolol is a β 1 -selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. Methods and Results —Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective β 2 -adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of β 2 - but not β 1 -adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca 2+ concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. Conclusions —In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial β 2 -adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca 2+ ] i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.

SUMMARY OM-89 is a bacterial (Escherichia coli) extract used for oral administration in the treatment of RA. Given the evidence that immunity to bacterial heat shock antigens plays a critical role in the immunomodulation of arthritis and possibly inflammation in general, the purpose of the present studies was to evaluate the presence and immunogenicity of hsp in OM-89. Furthermore, we studied the effects of OM-89 in an experimental arthritis, where hsp are known to have a critical significance in disease development. In rats immunization with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunization with hsp antigens was also found to induce T cell reactivity specific for OM-89. Based on this and the antigen specificity analysis of specific T cell lines, hsp70 (DnaK) turned out to be one of the major immunogenic constituents of OM-89. Parenteral immunization with OM-89 was found to reduce resistance to adjuvant arthritis (AA), whereas oral administration was found to protect against AA. Given the arthritis-inhibitory effect of oral OM-89 in AA, it is possible that peripheral tolerance is induced at the level of regulatory T cells with specificity for hsp. This may also constitute a mode of action for OM-89 as an arthritis-suppressive oral drug.

IgA nephropathy (IgAN) is the commonest cause of glomerulonephritis and clinical exacerbation of IgAN is frequently associated with mucosal infection. T-cell receptor gamma delta (TCR gamma delta+) cells are increased in both the circulation and in renal biopsies of patients with progressive IgAN. We examined the hypothesis that specific peptides within the 65,000 MW heat-shock protein (hsp) might stimulate TCR gamma delta cells and play a part in the immunopathogenesis of IgAN. We studied T-cell proliferative responses stimulated by overlapping peptides derived from the sequence of mycobacterial 65,000 MW hsp. Three T-cell epitopes have been identified (peptides 51-65, 71-85 and 281-295). The three peptides have a synergistic effect and they stimulate significantly higher proliferation of T cells in patients with IgAN than in disease or healthy controls. This response was inhibited by monoclonal antibodies (mAb) to TCR gamma delta+ and human leucocyte antigen (HLA) class I, but not by mAb to HLA class II. The involvement of TCR gamma delta+ cells was confirmed by up-regulation of the proportion of TCR gamma delta+ cells when stimulated with the three specific peptides. We suggest that IgAN might be associated with mucosal infection by a variety of micro-organisms and that peptides within the microbial hsp cross-react with the homologous human hsp which may stimulate TCR gamma delta+ cells and play a part in the pathogenesis of IgAN.

Background The mechanisms of the established atheroprotective effects of estrogen have not been entirely clarified. Recent data suggest that agents that hasten the recovery of the endothelium after denuding injury will deter the development of neointimal lesions. Because estrogen has been shown to exert angiogenic effects in vitro and in vivo, we performed a series of experiments to evaluate whether estrogen was capable of accelerating reendothelialization. Methods and Results Ovariectomized Sprague-Dawley rats received estrogen replacement therapy in the form of subcutaneously implanted pellets designed to release 1.5 or 5.0 mg 17β-estradiol over 30 days. Deendothelializing balloon injury was performed 1 week after pellet implantation, and animals were euthanatized after 1 week for evaluation of reendothelialization (Evans blue staining) or 2 weeks for evaluation of reendothelialization and neointimal formation. At both time points, the use of estradiol caused a dose-dependent increase in reendothelialization, which was measured as absolute area and percentage of area that is reendothelialized. Estradiol accelerated functional endothelial recovery, manifested as an increase in nitric oxide production. Neointimal thickening was also shown to be inhibited in a dose-dependent fashion. Conclusions Estrogen accelerates functional endothelial recovery after barotraumatic deendothelializing injury. These findings, along with the recent demonstration of estrogen receptor expression by endothelial cells, suggest that the antiatherogenic action of estrogen may be mediated in part through direct effects on endothelial cells.

Radio receivers should be robust to large out-of-band blockers with small degradation in their sensitivity. N-path mixers can be used as mixer-first receivers [1] with good linearity and RF filtering [2]. However, 1/f noise calls for large active device sizes for IF circuits and high power consumption. The 1/f noise issue can be relaxed by having RF gain. However, to avoid desensitization by large out-of-band blockers, a bandpass filter (BPF) with sharp cut-off frequency is required in front of the RF amplifiers. gm-C BPFs suffer from tight tradeoffs among DR, power consumption, Q and fc. Also, on-chip Q-enhanced LC BPFs [3] are not suitable due to low DR, large area and non-tunability. Therefore, bulky and non-tunable SAW filters are used. N-path BPFs offer high Q while their center frequency is tuned by the clock frequency [2]. Compared to gm-C filters, this technique decouples the required Q from the DR. The 4-path filter in [4] has only 2nd-order filtering and limited rejection. The order and rejection of N-path BPFs can be increased by cascading [5], but this renders a “round‿ passband shape. The 4th-order 4-path BPF in [6] has a “flat‿ passband shape and high rejection but a high NF. This work solves the noise issue of [6] while achieving the same out-of-band linearity and adding 25dB of voltage gain to relax the noise requirement of the subsequent stages.

A pathogenic role for self-reactive cells against the stress protein Hsp60 has been proposed as one of the events leading to autoimmune destruction of pancreatic beta cells in the diabetes of nonobese diabetic (NOD) mice. To examine this hypothesis, we generated transgenic NOD mice carrying a murine Hsp60 transgene driven by the H-2E alpha class II promoter. This would be expected to direct expression of the transgene to antigen-presenting cells including those in the thymus and so induce immunological tolerance by deletion. Detailed analysis of Hsp60 expression revealed that the endogenous gene is itself expressed strongly in thymic medullary epithelium (and weakly in cortex) yet fails to induce tolerance. Transgenic mice with retargeted Hsp60 showed overexpression of the gene in thymic cortical epithelium and in bone marrow-derived cells. Analysis of spontaneous T-cell responses to a panel of self and heterologous Hsp60 antigens showed that tolerance to the protein had not been induced, although responses to an immunodominant 437-460 epitope implicated in disease were suppressed, probably indicating an epitope shift. This correlated with changes in disease susceptibility: insulitis in transgenic mice was substantially reduced so that pathology rarely progressed beyond periislet infiltration. This was reflected in a substantial reduction in hyperglycemia and disease. These data indicate that T cells specific for some epitopes of murine Hsp60 are likely to be involved in the islet-cell destruction that occurs in NOD mice.

Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors. Our previous studies showed increased expression of human hsp60 on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis. To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shock human umbilical vein endothelial cells. Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells. High levels of hsp60 mRNA expression in endothelial cells were found between 4 and 12 h after 30 min treatment at 42 degrees C. In immunofluorescence tests, positive staining of heat-stressed endothelial cells was observed not only in the cytoplasm but also on the cell surface. Furthermore, only heat-stressed, but not untreated, Cr-labeled endothelial cells were lysed by anti-hsp65/60 Ab in the presence of complement (complement-mediated cytotoxicity) or peripheral blood mononuclear cells (antibody-dependent cellular cytotoxicity). Control Abs, including human anti-hsp65/60 low titer antiserum, human Ig fraction deprived of hsp65/60 Ab, and mAbs to Factor VIII, alpha-actin, hsp70, and CD3 showed no cytotoxic effect. In conclusion, human serum anti-hsp65 antibodies act as autoantibodies reacting with hsp60 on stressed endothelial cells and are able to mediate endothelial cytotoxicity. Thus, a humoral immune reaction to hsp60 may play an important role in the pathogenesis of atherosclerosis.

Lewis rats are susceptible to several forms of experimental arthritis-induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II-restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell-mediated regulation of inflammation.

Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.

—A widely tunable 4th order BPF based on the subtraction of two 2nd order 4-path passive-mixer filters with slightly different center frequencies is proposed. The center frequency of each 4-path filter is slightly shifted relative to its clock frequency (one upward and the other one downward) by a gm-C technique. Capacitive splitting of the input signal is used to reduce the mutual loading of the two 4-path BPFs and increase their quality factors. The filter is tunable from 0.4 GHz to 1.2 GHz with approximately constant bandwidth of 21 MHz. The in-band 1-dB compression point of the filter is -4.4 dBm while the in-band IIP3 of the filter is +9 dBm and the out of band IIP3 is +29 dBm (f= +50 MHz). The ultimate rejection of the filter is >55 dB and the NF of the filter is 10 dB. The static and dynamic current consumption of the filter are 2.8 mA from 2.5 V and 12 mA from 1.2 V, respectively (at 1 GHz). The LO leakage power to the input port is

Recent results suggested that a large subset of heat shock protein HSP-60 reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common HSP. However, the experimental evidence supporting this idea involved in vitro peptide responses of gamma delta T-cell hybridomas generated from unprimed spleen cells. Here, we report an attempt to elicit a gamma delta T-cell response in vivo by stimulation of adult C57BL/10 mice with HSP-60 or an HSP-60-derived peptide fragment comprising amino acids 180-196 of mycobacterial HSP-60. Whereas no gamma delta T-cell response was detectable in mice injected with the intact protein, stimulation with the peptide altered the reactive gamma delta T-cell population in vivo. These changes were detected among hybridomas generated with cells restimulated in vitro and included a large increase in hybridizable gamma delta T cells, a nearly maximal increase in the relative frequency of HSP-60-reactive cells, and structural changes in expressed T-cell receptors of HSP-60-reactive cells. Interestingly, we failed to elicit a detectable alpha beta T-cell response to the particular peptide stimulatory for gamma delta T cells, although at least three other HSP-60 epitopes were recognized. Our data show that normal gamma delta T cells can respond in vivo to small peptide antigens. The gamma delta T-cell response to the HSP-60-derived peptide studied here is apparently independent of antigen-specific alpha beta T-cell reactivity.

A frequency compensation technique for MOS audio power amplifiers is presented that allows the frequency compensation capacitors around the power transistors to be smaller than the circuit parasitics without power or stability penalty. Stability is analysed by inspecting the output impedance of the closed loop amplifier, instead of the traditional open-loop gain. By degenerating the gain of the penultimate stage, the output impedance is shaped such that the stability of the audio amplifier is guaranteed for complex loudspeaker loads. The realized amplifier features a THD of 0.005% @ (1 kHz, 10 W), an SNR of 110 dB(A), and stable operation for any passive load up to 50 nF.

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Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin-dependent diabetes mellitus of NOD mice, we studied the specificities of helper T-cell clones capable of causing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence of the human variant of the 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cell vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes.

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Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice. Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp70) had no effect on the development of diabetes.

Fundulopanchax kamdemi, new species (Figs. 2-4) Aphyosemion sp. 01 (cf. A. cameronense) ��(McGregor Reid, 1989: 27) Aphyosemion sp. (KORUP 01) (Kamdem Toham, 1992: 6-10) Fundulopanchax sp. aff. ndianus (Wildekamp, 1996: 193-194) Aphyosemion aff. amieti (Huber, 1996: 323) Fundulopanchax aff. ndianus (Huber, 2000: 472) Holotype. MRAC A2-003-P -0001, male, 50.0 mm SL; Cameroon: Korup National Park, small pools under forest cover; 05��05���N08��52���E; G. Chiambeng, 12 January 2001. Paratypes. MRAC A2-003-P -0002-0003, two males, 43.1-46.1 mm SL; same data as holotype. - MRAC 92-144-P -0384-0386, 2 males, 40.5-45.7 mm SL and female, 47.3 mm SL; Cameroon: Korup National Park: west of science camp; 05��05���N08��52���E; A. Kamdem Toham, 19 December 1992. - ZFMK 39900-39917, 7 males, 38.3-54.4 mm SL and 11 females, 31.9-51.6 mm SL; Cameroon: Korup National Park, small forest stream emptying in swampy pools; 05��00���20������N08��47���01������E; C. Akum, December2001. - ZFMK 39918-39931, juvenile specimens, not measured; Cameroon: Korup National Park, small forest stream emptying in swampy pools; 05��00���20������N08��47���01������E; C. Akum, December 2001, same data as ZFMK specimens. - IRAD Research Station, Batoke, Limbe, Cameroon, four specimens, two males and two females; Cameroon: Korup National Park, small forest stream emptying in swampy pools; C. Akum, December 2001. Additional material. Fundulopanchax kamdemi Fundulopanchax ndianus (Scheel, 1968), MRAC 73-39-P -1649-1660 (paratypes); Nigeria, near Osomba, H.S. Clausen and J.J. Scheel, 1961. MRAC 84-51-P -27-28; Nigeria, 88 km NE on MCC road. (female in this lot = Aphyosemion calliurum). MRAC 84-51-P -162-163; Nigeria, Nsan village, appr. 40 km N. of Calabar (female in this lot = Aphyosemion calliurum). Fundulopanchax mirabilis MRAC A2-003-P -0004-0008; Cameroon, Korup National Park; G. Chiambeng, 12 January 2001. Uncatalogued material in the collection of the second author, used for DNA studies, of the following species: F. amieti, F. arnoldi, F. avichang, F. deltaensis, F. fallax, F. gularis, F. ndianus, F. puerzli, F. rubrolabialis, F. sjoestedti, F. spoorenbergi, F. traudeae and F. walkeri. Diagnosis. Fundulopanchax kamdemi shares with all other Fundulopanchax species except those of the subgenus Paludopanchax 16 or more scales around the caudal peduncle versus 12 in Aphyosemion s.l. It is distinguished from all Aphyosemion species and many Fundulopanchax except F. arnoldi, F. deltaensis, F. gularis, F. kribianus, F. ndianus, F. robertsoni, F. rubrolabialis, F. schwoiseri, F. sjoestedti, and F. walkeri by the high number of dorsal (15-18) and anal fin rays (16-19) (according to the descriptions and data in Huber 2000). It is distinguished from all Fundulopanchax species by its unique male coloration of a red longitudinal band on the middle of the sides versus no red band, with the exception of F. amieti, F. avichang, F. deltaensis and some individual specimens of F. ndianus, F. puerzli and F. spoorenbergi. It is distinguished from all Fundulopanchax species, except F. ndianus and some specimens of F. puerzli, by a red ventral band from the pelvic fins to the lower caudal fin base. F. kamdemi is distinguished from the latter species and most other Fundulopanchax except F. spoorenbergi by the coloration of the unpaired fins as given below. Females can be distinguished from its congeners by the presence of an orange-red margin at the dorsal fin and a narrow red band at the base of the anal fin. Description. See Figures 2-4 for general appearance and Table 1 for morphometric data of the type specimens. Fundulopanchax of large size. Dorsal-fin origin distinctly behind mid-length of body and just behind anal fin origin. Dorsal fin 15-18 rays, anal fin 16-19 rays. Scales on mid-longitudinal series 34-38 plus 3 or 4 on caudal fin base, most with shallow pit in centre, not connected to underlying neuromast system. Transverse rows of scales above pelvic fin 10-11; scale rows around caudal peduncle 16. Supra-orbital squamation G- pattern, with two H-scales. Anterior and central cephalic neuromast systems separated, consisting of two shallow grooves, lined with low lobes. Posterior cephalic neuromast system, consisting of 3 neuromasts, in an open curved groove, with both ends angled to approximately 90��. Preopercular neuromast system tubular with six exposing pores. Males. Up to 73.0 mm SL. Body laterally compressed. Dorsal profile nearly straight. No distinct transition between head and body. Dorsal and anal fin trapezoid, pointed at distal end. Dorsal and anal fin rays slightly projecting from fin membrane. Both fins covered with a thin layer of epidermal tissue. No visible papillae on dorsal and anal fin rays. Opercular membrane slightly projecting posteriorly, distal edge smooth or only slightly wrinkled. Many small and hair like, probably epithelial, papillae at the distal margin of the scales in the mid-longitudinal and lower scale row at caudal peduncle, beginning above the anal fin. This phenomena was also described for F. ndianus (Scheel, 1968), F. puerzli (Radda & Scheel, 1974) and F. amieti (Radda, 1976). Scheel (1968) described it ctenoid spines at some scales. In the descriptions of F. puerzli and F. amieti these where named ctenii or 'Kontaktorgane' (contact organs) (Radda, 1976; Radda & Scheel, 1974). Similar structures were observed by us in F. fallax (Ahl, 1935) and F. traudeae (Radda, 1971). These hair like structures seem to be of a similar origin as the ctenii on the distal margin of the scales on the species mentioned above. They can vary in appearance from short and stout (F. ndianus) to long and hair like (F. fallax, F. amieti and F. kamdemi sp. nov.). It is to note that not all individuals of a species show these structures (Scheel, 1990, and own observation), which makes it difficult to draw conclusions about its distribution in the different Fundulopanchax species groups. The origin and function of these structures is not known and will be subject of a future study. It is thought that they play a role during spawning like the fin and scale papillae in the genus Nothobranchius. Females. Up to 61.7 mm SL. Body less laterally compressed and deeper than male. Dorsal profile straight. Dorsal and anal fin trapezoid, tip rounded. Caudal fin rounded. No epidermal tissue present on dorsal and anal fins. Opercular membrane not projecting posteriorly. No papillae at the body scales. Coloration. Males (Fig.2) Live specimen. Dorsal brown to red-brown. Sides dominated by a bright red longitudinal band, extending from upper opercle junction to caudal fin base. On anterior part of body this band may be interrupted, as in Fig. 2. On anterior part of sides a short red band or series of red spots usually below longitudinal band. Body coloration above red band reflective green to blue-green. Below band reflective light blue. Red-brown of dorsal area usually separated from the green on sides by a series of red spots. A further red band, following lower body profile, from pelvic fins to caudal fin base. Upper part of the head brown, opercle reflective green. Throat and lower part of the head light blue. On opercle three parallel oblique red streaks. Under eye a red band and a further red band just below the lower lip. Lobes lining supra-orbital neuromast systems red. Lower part of dorsal fin reflective green-blue with series of red dots at base. Dorsal fin margin yellow-green separated from green-blue by a red band. Anal fin light blue. At the base a red band. Anal fin margin dark red. Caudal fin reflective green-blue to light blue. On central part an irregular pattern of red flame-like stripes parallel to the rays. One or two flames originating from end of red body band. Upper caudal fin margin yellow-green, separated from central part by a red band. Upper caudal fin margin ending in a long extension. Lower edge of caudal fin dark red, separated from central part by a white, light blue or in some specimen yellow band. Pelvic fins light blue with red margin. Pectoral fins transparent light blue with red sub-terminal band and light blue edge. Females (Fig. 3) Live specimen. Body coloration generally grey-brown to yellow-brown. Upper part of sides with light green cast. Lower part of sides with light blue. On middle of sides a series of red spots. On anterior part of body a second series of red spots below it. Lower part of the sides, ventrum and throat pale orange. Lower half of dorsal fin reflective green with some red spots at base. Upper half of dorsal fin orangered. Basal part of anal fin light blue with red horizontal band. Remaining part of anal fin pale yellow with red edge. Caudal fin transparent pale green-yellow. Upper part and upper edge of caudal fin orange-red with some red spots at base. Pelvic fins orange. Pectoral fins hyaline. Preserved in ethanol. Males (Fig. 4). Pale white-yellow band distinctly present on mid-lateral body. Longitudinal band separates dark grey-brown dorso-lateral part of body from yellow-grey ventro-lateral part. Anterior part of mid-lateral band usually consisting of two parallel horizontal lines, in some specimens represented by series of dots. The two parallel horizontal lines fuse to one band above middle of anal fin base. Upper part of head grey-brown, lower part and throat pale yellow. Opercle with 2 to 3 parallel pale white-yellow oblique bars. Dorsal fin semi-transparent grey. A purple band runs from the middle of the first dorsal fin ray to the pointed distal end separating the light grey margin from the rest of the fin. In most specimens the purple band is forked, its lower branch reaching distal dorsal fin base. At base of dorsal fin two or three purple spots. Anal fin with pale yellow band at base, followed by a pale grey part and black to dark brown margin. In some individuals the black to dark brown band may be followed by a grey-white margin. Caudal fin purplebrown. The white-yellow body band continues in the caudal fin and ends in the upper extension. Upper caudal fin margin as dorsal fin margin. From lower part of caudal fin base a pale yellow band runs obliquely upward to about half-way along the fin. From there it runs downward, parallel to fin rays, to the lower caudal fin extension. Fin part below band purple-brown followed by grey-white submargin and black to dark brown margin. Females. Uniform grey-brown on body and sides. Vague band of light grey-yellow spots running from the upper opercle to the caudal fin base. All fins uniformly semi-transparent grey. Distribution. Fundulopanchax kamdemi is known from a limited number of localities, all within the Korup National Park. It was mentioned as Aphyosemion sp. 01 (cf. A. cameronense) in McGregor Reid (1989) from the Akpa-Yafe River and the upper Ndian River, in southern Korup only. This was confirmed by comparing the specimens, deposited by Kamdem Toham, as Aphyosemion sp. (KORUP 01) in the collection of the Museum for Central Africa, Tervuren, Belgium. He found them in the swampy parts that edge the small creeks under the cover of the forest in the southern Korup close to the Science camp (Kamdem Toham, 1992). Additionally the three collection localities, studied by the first author, confirmed the presence of the new species in the southern part of the Park only. Presence in the adjacent Oban National Park at the Nigerian side of the border may be possible but could not be confirmed as no collections of cyprinodontiform fishes are known from there. Other cyprinodontiform species found in sympatry with F. kamdemi are F. marmoratus, Epiplatys infrafasciatus (G��nther, 1866) (= E. sexfasciatus in McGregor Reid, 1989 and Kamdem Toham, 1992), Aphyosemion (Chromaphyosemion) bivittatum, A. calliurum and Aplocheilichthys spilauchen. All have a larger distribution and are also known from localities outside the park. Ecology. According to Kamdem Toham (1992) the species generally lives in the shallow swampy pools at the edge of small creeks under forest cover. These pools, up to 35 cm in depth, are partly covered with a layer of fallen leaves under which the fishes in the pools take cover. Additional ecological information was collected by the first author. The presence of F. kamdemi was also recorded from small forest streams connected with swampy pools. Streams and pools are heavily shaded by peripheral vegetation and their bottom is covered by decaying leaves, branches and logs. Both the streams and pools contain clear water during the rainy season and brown tinged during the dry season. Water depths ranged during the seasons from 2 to 48.3 cm. Water temperature was measured between 20.9 and 23.8�� C, the pH between 5.0 and 7.5. Water hardness varied between 0.6 and 1 DH, conductivity between 10 and 21 ��S/cm and the total dissolved oxygen between 0.9 and 5.5 mg/l. Stomach contents of F. kamdemi indicated an insectivorous behavior and consisted of ants, crickets, beetles, spiders and cockroaches, with a predominance of ants. In the wild, maturity was observed in the months of November to January. These shallow pools dry out periodically, suggesting an annual mode of reproduction. The first observations on the breeding biology of F. kamdemi were carried out in captivity by K.-H. L��ke, Bochum and W. Eigelshofen, Sprockh��vel, both Germany indicate that its annual mode of reproduction is facultative. Embryological development has not been studied. The cyprinodontiform fish fauna of the Korup National park. The cyprinodont fauna of the Korup National Park and its surroundings is diverse. The collections made by McGregor Reid and Kamdem Toham (specimens collected by the latter are in the collection of the Museum for Central Africa, Tervuren, Belgium) included species of five different genera (e.g. Epiplatys, Aplocheilichthys, Aphyosemion, Fundulopanchax and Procatopus). McGregor Reid (1989) distinguished, based on male coloration, four different phenotypes of the genus Procatopus and Kamdem Toham (1992) distinguished two. Based on Van der Zee, Woeltjes and Wildekamp (in press) two Procatopus species occur in the vicinity of the Korup National Park, P. aberrans Ahl, 1927 and P. similis Ahl, 1927. Both are variable in male coloration at the population level, but P. aberrans generally is restricted to the soils of basement crystalline origin and P. similis to soils of sedimentary origin. In the areas surrounding the park, in Nigeria as well as in Cameroon, the occurrence of Aphyosemion calliurum, A. (Chromaphyosemion) cf. splendopleure (Br��ning, 1929), F. sjoestedti (L��nnberg, 1895), F. marmoratus, F. scheeli (Radda, 1970), F. ndianus (Berkenkamp, 1976) and E. grahami (Boulenger, 1911), have been demonstrated (Huber, 2000; Radda & P��rzl, 1982; Wildekamp, 1993, 1996). Etymology. Named for Andre Kamdem Toham of the World Wildlife Fund���s Central African Rainforest Project (CARPE) and a collector of this new species. Discussion The tubular preopercular neuromast system of this new species has six exposing pores, indicating that it is a representative of the Fundulopanchax / Aphyosemion-group within the family Nothobranchiidae of the cyprinodontiform suborder Aplocheiloidei. The additional diagnostic characters, with the exception of otolith morphology which was not studied, given in Van der Zee & Wildekamp (1994), all fit to F. kamdemi which is therefore attributed to the genus Fundulopanchax. The male color pattern of the adult F. kamdemi is dominated by the red longitudinal band on the sides. This character is shared by two other Fundulopanchax species, F. amieti (see Seegers, 1997) and F. avichang Malumbres & Castello, 2001, and can also be found in most male specimens of the Dibamba, Cameroon, population of F. puerzli (see Amiet, 1987) and individuals of F. spoorenbergi. In F. kamdemi a second red band runs parallel to the lower body profile, a character shared only by F. ndianus. With the exception of F. avichang all species mentioned share all distinguishing characters for the genus Fundulopanchax mentioned in Van der Zee and Wildekamp (1994) and all are robust species of large (= 55 mm SL) size. Fundulopanchax avichang differs from the above mentioned species by its small size (less than 40 mm SL), low number of circumcaudal scales (12-13 vs. 16) and elliptical caudal fin in males (vs. extensions at the upper and lower corner). The second red line on the lower body profile is not a unique derived character since, beside its presence in Fundulopanchax (e.g. F. avichang and F. amieti), it is found in some species of the related genus Aphyosemion (e.g. A. ferranti (Boulenger, 1910), A. labarrei Poll, 1951, A. louessense (Pellegrin, 1931), and populations of A. cameronense (Boulenger, 1903))., Published as part of Christian Akum, Rainer Sonnenberg, Jouke R. van der Zee & Rudolf H. Wildekamp, 2007, Fundulopanchax kamdemi (Cyprinodontiformes: Nothobranchiidae) a new species from Korup National Park, western Cameroon., pp. 41-49 in Zootaxa 1532 on pages 43-48

[[Fundulopanchax Myers]] Fundulopanchax was originally described by Myers (1924) as a subgenus in Aphyosemion Myers, 1924 but his diagnostic characters were not adopted in subsequent revisions. On the basis of two synapomorphic characters, Parenti (1981) recognized Fundulopanchax as a monophyletic sister group to Nothobranchius Peters, 1868 and consequently designated Fundulopanchax as a genus. Van der Zee and Wildekamp (1994) accepted the generic status of Fundulopanchax, but the value of Parenti���s (1981) diagnostic characters was disputed. Additionally, they found four diagnostic characters for the genus Fundulopanchax: chorionic structure (puncti), anal-fin skeleton (lateral processes on anal radialia reduced), otolith morphology and caudal peduncle squamation (16 or more scales, except Paludopanchax Radda, 1977) (see Van der Zee & Wildekamp, 1994). Murphy and Collier (1999), using mitochondrial DNA sequences, corroborated the generic status of Fundulopanchax, but they did not, in most cases, find correlation between the species groups as proposed by Radda (1977) on morphological arguments. In Murphy and Collier (1999), Aphyosemion is the sister group to Fundulopanchax, whereas in a second molecular genetic study, based on more limited material and focused on the evolution of diapause in New World Rivulidae, by Hrbek and Larson (1999), Aphyosemion is the sister group to a clade comprised of Fundulopanchax and Nothobranchius (Fig. 1). Morphological characters discriminate between Aphyosemion and Fundulopanchax but it should be the subject of a future study if they are apomorphies of a monophyletic group Fundulopanchax, or if in a broader study with the remaining African Nothobranchiidae, they will be assessed as plesiomorphic characters. The distribution of Fundulopanchax ranges from C��te d���Ivoire to Equatorial Guinea with the highest species diversity in southeastern Nigeria and adjacent southwestern Cameroon (species numbers and distribution see Huber, 2000; Wildekamp, 1996)., Published as part of Christian Akum, Rainer Sonnenberg, Jouke R. van der Zee & Rudolf H. Wildekamp, 2007, Fundulopanchax kamdemi (Cyprinodontiformes: Nothobranchiidae) a new species from Korup National Park, western Cameroon., pp. 41-49 in Zootaxa 1532 on pages 41-42

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The significance of immune responses to certain heat shock proteins (HSPs) that develop in virtually all inflammatory diseases is only now becoming clear. In experimental models, HSPs prevent or arrest inflammatory damage, and initial clinical trials in chronic inflammatory disease have shown HSP peptides to promote production of anti-inflammatory cytokines-indicating immunoregulatory potential. HSPs are ubiquitous self-antigens that are highly expressed in inflamed tissues. The prokaryotic homologous proteins, present in every bacterial species, are dominantly immunogenic. This is striking, especially as these proteins have large areas of sequence homologies with the host (mammalian) counterparts. In several experimental models of autoimmune diseases, immunisation with bacterial HSPs inhibited disease development, as did oral/nasal administration. Based on the experimental evidence so far, it is tempting to speculate that: firstly, exposure to homologues of these self-antigens, as present in, for instance, the bacterial intestinal flora, has a decisive impact on the regulation of self-tolerance at the level of T cells; and secondly, such proteins or their derivative peptides may have a role in an antigen specific immunotherapy approach involving modulation of relevant T cells, without the immediate necessity of defining disease specific autoantigens. Recent findings in experimental asthma and atherosclerosis have indicated that the field of application of such immunotherapy can be broader than just autoimmunity.

Background—Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis.Methods and Results—Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor−/−mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%;PP+CD25+Foxp3+cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-β production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis.Conclusions—OxLDL-specific T cells, present in LDL receptor−/−mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+CD25+Foxp3+regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Immunisation with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunisation with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp601 and later for mycobacterial hsp70.2,3 Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunisation with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60.4 Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. References Anderton SM, van der Zee R, Prakken B, Noordzij A, van Eden W. Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J Exp Med. 1995;181:943–52 Tanaka S, Kimura Y, Mitani A, Yamamoto G, Nishimura H, Spallek R, Singh M, Noguchi T, Yoshikai Y. Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis. J Immunol. 1999;163:5560–65 Wendling U, Paul L, van der Zee R, Prakken B, Singh M, van Eden W. A conserved mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the mammalian self-hsp70 homologue. J Immunol. 2000;164:2711–17 Paul AG, van Der Zee R, Taams LS, van Eden W. A self-hsp60 peptide acts as a partial agonist inducing expression of B7-2 on mycobacterial hsp60-specific T cells: a possible mechanism for inhibitory T cell regulation of adjuvant arthritis? Int Immunol. 2000;12:1041–50

Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp600 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10.

Oral administration of antigen prior to disease induction has been shown to induce peripheral tolerance in several experimental autoimmune diseases. However, the clinical benefit of pretreatment with antigens is limited. The aim of this study was to investigate whether adjuvant-induced arthritis (AIA) could be treated by oral administration of mycobacterial heat-shock protein 65 (Hsp65) during ongoing disease.AIA was induced in Lewis rats by immunization with Mycobacterium tuberculosis in Freund's incomplete adjuvant. Oral feeding of Hsp65 in the presence or absence of soybean trypsin inhibitor (SBTI) was started on day 11 after immunization. Arthritis was monitored visually, and joint pathology was examined radiologically.Oral treatment with Hsp65 during ongoing disease significantly reduced the activity of AIA. However, treatment with Hsp65 was only successful when SBTI was coadministered to prevent breakdown of the Hsp65. The beneficial effect of Hsp65/SBTI treatment during AIA was also represented by a clear reduction of articular destruction, as visualized by radiography. Moreover, feeding Hsp65/SBTI resulted in a lower number of both spleen and mesenteric lymph node (MLN) cells expressing the costimulatory molecule CD80 (B7-1). The number of cells expressing CD86 (B7-2) was not altered. Furthermore, MLN cells from AIA animals treated with Hsp65/SBTI contained a lower number of T cells expressing the activation marker CD134 (Ox-40). In addition, treatment with Hsp65/ SBTI was accompanied by an increased proliferative response of spleen cells to the Hsp65 antigen in vitro. Moreover, Hsp65/SBTI-treated rats showed less Hsp65-specific interferon-gamma and increased production of interleukin-10.Ongoing AIA activity can be reduced by oral administration of Hsp65 only when protein breakdown in the gastrointestinal tract is inhibited.

SUMMARYT cell-mediated immunity is thought to play an important role in the pathogenesis of WG. In previous studies a minority of WG patients as well as some healthy controls showed in vitro proliferation of their peripheral blood mononuclear cells (PBMC) to PR3, the main autoantigen in WG. The relevant peptides responsible for this in vitro proliferation have not been identified. In order to define immunogenic peptides, PBMC of 13 WG patients in remission and 10 healthy controls were tested for proliferation to linear peptides of PR3 and to whole PR3. Fifty overlapping peptides spanning the whole PR3 sequence were synthesized. Peptides were tested in pools of five peptides and as single peptide. PBMC of two WG patients and one healthy control proliferated to whole PR3 and to peptide pools. In addition, 10 WG patients and eight healthy controls that did not proliferate to whole PR3 did proliferate to pools of PR3 peptides. Although more WG patients tended to react to particular peptide pools, no significant difference was seen between lymphocyte proliferation to PR3 peptides of WG patients and that of healthy controls. The pools of peptides recognized were mainly located at the N- and C-terminus of PR3. No correlation was observed between HLA type and proliferation on particular peptide pools. No proliferation of PBMC was observed to single peptides. In conclusion, T cells of WG patients proliferate in vitro more frequently to PR3 peptides than to the whole PR3 protein. Peptides derived from the signal sequence, the propeptide or peptides located at the C-terminus of PR3 induce highest levels of proliferation. No specific PR3 sequence could be identified that was preferentially recognized by PBMC of WG patients compared with controls.

The adhesion integrin alpha(v)beta3 is expressed by both activated endothelial cells (ECs) and smooth muscle cells (SMCs). Peptide and antibody antagonists of alpha(v)beta3 have been shown to block angiogenesis by initiating unscheduled programmed cell death of proliferating ECs. The present study was designed to determine if antagonism of alpha(v)beta3 immediately following balloon injury might similarly lead to programmed cell death among activated SMCs, and thereby inhibit intimal thickening. LM609, a monoclonal antibody antagonist of alpha(v)beta3, was administered locally and/or systemically immediately after balloon angioplasty in a rabbit model of vascular injury. Immunohistochemical studies documented that LM609, even when administered systemically, localized to sites of vascular injury. LM609 administered immediately following balloon injury of the external iliac artery markedly reduced intimal thickening at 2 and 4 wk post-injury. Apoptosis was abundant where balloon injury resulted in expression of alpha(v)beta3. At both 2 and 4 wk, re-endothelialization at the site of balloon injury was not retarded in LM609-treated rabbits versus controls. Thus, blockade of alpha(v)beta3 inhibits intimal thickening when administered immediately following balloon injury. This favorable impact on neointimal thickening is associated with apoptosis of activated SMCs expressing alpha(v)beta3. These findings may explain the reduction in restenosis observed clinically following beta3 integrin blockade.

Objective. To determine whether T lymphocyte reactivity to endogenous human hsp60 plays a regulatory role in the course of oligoarticular juvenile rheumatoid arthritis (JRA). Methods. A prospective, longitudinal study of T cell reactivity to HSP in 15 patients with newly diagnosed HLA-B27 negative oligoarticular JRA was performed. Results were compared with those in a group of 20 patients with newly diagnosed polyarticular or systemic JRA or with acute arthritis caused by other systemic diseases or viral infections, as well as with those in a group of 9 healthy control subjects. Results. In 86% of the patients with oligoarticular JRA (13 of 15), significant T lymphocyte proliferative responses to hsp60 were found in peripheral blood mononuclear cells and/or synovial fluid mononuclear cells within 3 months after the onset of arthritis. Only 5% of the patients in the rheumatologic disease control group (1 of 20) showed such positivity. All patients with oligoarticular JRA and positive responses to human hsp60 developed a remission of their disease within 12 weeks. During this period of remission, blood samples were taken from 8 patients and showed significantly lower and even negative responses to hsp60, compared with active disease, when all 8 patients had good responses. Conclusion. The results show that significant proliferative responses to human hsp60 can be found early in the course of oligoarticular JRA. Furthermore, these responses correlate with disease activity in such a manner that T cell reactivity to human hsp60 seems to be associated with disease remission.

SUMMARYT cell epitopes of the 65-kD heat shock protein (hsp) were investigated in patients with recurrent oral ulcers (ROU). Peripheral blood mononuclear cells were stimulated with overlapping synthetic peptide (15ers), derived from the sequence of the 65-kD hsp of Mycobacterium tuberculosis. Specific lymphoproliferative responses were stimulated only with peptide 91–105 in ROU, compared with healthy or disease controls (P < 0·01). This was confirmed by studying 760 short term cell lines generated with the 65-kD hsp and then stimulated with the peptides. The frequency of short term cells lines responding to peptide 91–105 in ROU was significantly greater than in healthy (P < 0.0001) or disease controls (P < 0·01). A comparative investigation with the homologous human 60-kD hsp peptide 116–130 also showed significantly greater lymphoproliferative responses in ROU than in healthy (P < 0·01) or disease controls (P < 0·001). The potential involvement of the T cell epitope 91–105 in the pathogenesis of ROU is supported by finding a significant increase in the lymphoproliferative responses stimulated with peptide 91–105 during the stage of ulceration, compared with remission in 9/11 patients studied sequentially (P < 0·05). The results suggest that oral ulceration might be initiated by the microbial hsp peptide 91–105 stimulating the mucosal Langerhans cells, which may generate autoreactive T cell clones primed to the homologous peptide 116–130.

Synovial fluid and peripheral blood mononuclear cell proliferative responses to the 60-kD human heat shock protein (HSP60) were studied in 23 patients with juvenile chronic arthritis (JCA) and 7 non-JCA control patients. All patients showed active arthritis at the time of study. The patients were divided into two groups according to the presence (group A) or absence (group B) of T lymphocyte reactivity to human HSP60. We show that reactivity to human HSP60 is primarily, though not exclusively, occurring in patients with a remitting course of disease, i.e., the subgroup of HLA-B27 negative JCA patients with an oligoarticular onset. Immunohistochemical analysis of HSP expression in synovial membranes showed a significantly higher intensity of staining in JCA patients than in non-JCA controls. The results suggest that, in accordance with the earlier observation made in experimental models, T lymphocyte reactivity to human HSP60 in this subgroup of JCA patients may be part of T cell regulatory mechanisms that control the development of arthritis.

In the epidermis, the major population of T lymphocytes expresses a T-cell receptor (TCR) with V gamma 5 and V delta 1 variable regions, which is unique to this tissue. Roberts et al and Ezquerra et al, also describe a minor population of gamma delta-TCR+ cells in the epidermis that expresses a V gamma 1/V delta 6 TCR. These cells are different from other epidermal T cells in that they "spontaneously" produce cytokines, a result thought to be due to autoreactivity. Over the past 5 years, our laboratory has produced V gamma 1/V delta 6+ T-cell hybridomas from many tissue sources. These spontaneously produce cytokines but also are activated by heat shock protein (HSP-60)-derived peptides. Ezquerra et al report that none of their V gamma 1/V delta 6+ epidermal T-cell lines derived from C3H/HeN mice respond to HSP-60. Of the 99 gamma delta-TCR+ hybridomas we have produced from epidermal T cells of C57BL/6 mice, only one expressed the V gamma 1/V delta 6 TCR. This hybridoma, 70BET-2.12, not only spontaneously produces cytokines, but, unlike the V gamma 1/V delta 6-TCR+ epidermal T cells of Ezquerra et al, it also responds to the whole HSP-60 protein and a 17-mer HSP-60 peptide from M. leprae, producing increased levels of interleukin-2 of up to approximately ten-fold above the spontaneous level. This shows that V gamma 1/V delta 6-TCR+ epidermal T cells can respond to HSP-60. To confirm that 70BET-2.12 expresses TCR genes similar to those of cells that have HSP-60 reactivity, V gamma 1-C gamma 4 and V delta 6-C delta cDNA were produced from RNA isolated from this hybridoma, amplified by the polymerase chain reaction, and sequenced. The gamma and delta TCR gene sequences were similar but not identical to previously published sequences of HSP-60-reactive cells from lymphoid and other organs. No explanation can be found for the discrepancy between our findings and those of others at the level of TCR expression such that other strain-specific factors might be responsible for HSP-60 reactivity.