Ben Min-Woo Illigens

Tolerance to Noninherited Maternal MHC Antigens in Mice

The phenomenon of tolerance to noninherited maternal Ags (NIMA) is poorly understood. To analyze the NIMA effect C57BL/6 (H-2b/b) males were mated with B6D2F1 (H-2b/d) females, whereby 50% of the offspring are H-2b/b mice that have been exposed to maternal H-2d alloantigens. Controls were H-2b/b offspring of C57BL/6 mothers, either inbred C57BL/6 mice or F1 backcross mice from breedings with H-2b/d fathers. We found that 57% of the H-2b/b offspring of semiallogeneic (H-2b/d) mothers accepted fully allogeneic DBA/2 (H-2d/d) heart grafts for >180 days, while similar transplants were all rejected by day 11 in controls (p < 0.0004). Foster nursing studies showed that both oral and in utero exposure to NIMA are required for this tolerogenic effect. An effect of NIMA was also found to extend the survival of skin grafts from a semiallogeneic donor (p < 0.02). Pretransplant analysis of splenocytes showed a 40–90% reduction of IL-2-, IL-5-, and IFN-γ-producing T cells responding to H-2d-expressing APC in NIMAd-exposed vs control mice. Injection of pregnant BALB/c-dm2 (H-2Ld-negative) female mice i.v. with H-2Ld61–80 peptide profoundly suppressed the offspring’s indirect pathway alloreactive CD4+ T cell response to H-2Ld. These results suggest that the natural exposure of the fetus and newborn to maternal cells and/or soluble MHC Ags suppresses NIMA-allospecific T cells of the offspring, predisposing to organ transplant tolerance in adult mice.

Sweat testing to evaluate autonomic function

Sudomotor dysfunction is common in many subtypes of neuropathy but is one of the earliest detectable neurophysiologic abnormalities in distal small fiber neuropathy. Clinical assessments of sudomotor function include thermoregulatory sweat testing (TST), quantitative sudomotor axon reflex testing (QSART), silicone impressions, the sympathetic skin response (SSR), the acetylcholine sweat-spot test and quantitative direct and indirect axon reflex testing (QDIRT). These testing techniques, when used in combination, can detect and localize pre- and postganglionic lesions, can provide early diagnosis of sudomotor dysfunction and can monitor disease progression or disease recovery. In this article, we describe many of the common clinical tests available for evaluation of sudomotor function with focus on the testing methodology and limitations while providing concrete examples of test results.

Quantification of sweat gland innervation A clinical–pathologic correlation

Objective: To evaluate a novel method to quantify the density of nerve fibers innervating sweat glands in healthy control and diabetic subjects, to compare the results to an unbiased stereologic technique, and to identify the relationship to standardized physical examination and patient-reported symptom scores. Methods: Thirty diabetic and 64 healthy subjects had skin biopsies performed at the distal leg and distal and proximal thigh. Nerve fibers innervating sweat glands, stained with PGP 9.5, were imaged by light microscopy. Sweat gland nerve fiber density (SGNFD) was quantified by manual morphometry. As a gold standard, three additional subjects had biopsies analyzed by confocal microscopy using unbiased stereologic quantification. Severity of neuropathy was measured by standardized instruments including the Neuropathy Impairment Score in the Lower Limb (NIS-LL) while symptoms were measured by the Michigan Neuropathy Screening Instrument. Results: Manual morphometry increased with unbiased stereology (r = 0.93, p < 0.01). Diabetic subjects had reduced SGNFD compared to controls at the distal leg (p < 0.001), distal thigh (p < 0.01), and proximal thigh (p < 0.05). The SGNFD at the distal leg of diabetic subjects decreased as the NIS-LL worsened (r = −0.89, p < 0.001) and was concordant with symptoms of reduced sweat production (p < 0.01). Conclusions: We describe a novel method to quantify the density of nerve fibers innervating sweat glands. The technique differentiates groups of patients with mild diabetic neuropathy from healthy control subjects and correlates with both physical examination scores and symptoms relevant to sudomotor dysfunction. This method provides a reliable structural measure of sweat gland innervation that complements the investigation of small fiber neuropathies.

Muscle engraftment of myogenic progenitor cells following intraarterial transplantation.

Cell‐based therapy continues to be a promising avenue for the treatment of Duchenne muscular dystrophy (DMD), an X‐linked skeletal muscle–wasting disease. Recently, we demonstrated that freshly isolated myogenic progenitors contained within the adult skeletal muscle side population (SP) can engraft into dystrophic fibers of nonirradiated mdx5cv mice after intravenous transplantation. Engraftment rates, however, have not been therapeutically significant, achieving at most 1% of skeletal muscle myofibers expressing protein from donor‐derived nuclei. To enhance the engraftment of transplanted myogenic progenitors, an intraarterial delivery method was adapted from a previously described procedure. Cultured, lentivirus‐transduced skeletal muscle SP cells, derived from mdx5cv mice, were transplanted into the femoral artery of noninjured mdx5cv mice. Based on the expression of microdystrophin or green fluorescent protein (GFP) transgenes in host muscle, sections of the recipient muscles exhibited 5%–8% of skeletal muscle fibers expressing donor‐derived transgenes. Further, donor muscle SP cells, which did not express any myogenic markers prior to transplant, expressed the satellite cell transcription factor, Pax7, and the muscle‐specific intermediate filament, desmin, after extravasation into host muscle. The expression of these muscle‐specific markers indicates that progenitors within the side population can differentiate along the myogenic lineage after intraarterial transplantation and extravasation into host muscle. Given that femoral artery catheterization is a common, safe clinical procedure and that the transplantation of cultured adult muscle progenitor cells has proven to be safe in mice, our data may represent a step toward the improvement of cell‐based therapies for DMD and other myogenic disorders. Muscle Nerve, 2006

Modulation of Tissue-Specific Immune Response to Cardiac Myosin Can Prolong Survival of Allogeneic Heart Transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4+ Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-γ-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient’s CD4+ T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.

Quantification of sudomotor innervation: A comparison of three methods

Peripheral sudomotor dysfunction is present in many peripheral neuropathies, but structural assessments of sudomotor fibers rarely occur. We evaluated 36 diabetic and 72 healthy control subjects who underwent detailed neurologic examinations and punch skin biopsies. Physical exam findings were quantified by neuropathy impairment score in the lower limb. Skin biopsies quantified intraepidermal nerve fiber density (IENFD) and sweat gland nerve fiber density (SGNFD) by a manual, automated, and semiquantitative method. The automated and manual SGNFD correlated with the IENFD at the same site (r = 0.62, P < 0.05 automated method, r = 0.67, P < 0.05 manual method). As neuropathy worsened, the SGNFD at the distal leg declined (automated counting r = −0.81, P < 0.001; manual counting r = −0.88, P < 0.001). The semiquantitative method displayed poor inter‐ and intrareviewer reliability and correlated poorly with standard neuropathy evaluation scores. Our results suggest that sudomotor fibers can be rapidly and reproducibly quantified, and results correlate well with physical exam findings. Muscle Nerve, 2010

The relative contribution of direct and indirect antigen recognition pathways to the alloresponse and graft rejection depends upon the nature of the transplant.

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.

QDIRT Quantitative direct and indirect test of sudomotor function

Objective:To develop a novel assessment of sudomotor function. Background:Postganglionic sudomotor function is currently evaluated using the quantitative sudomotor axon reflex test (QSART) or silicone impressions. We hypothesize that high-resolution digital photography has advanced sufficiently to allow quantitative direct and indirect reflex testing of sudomotor function (QDIRT) with spatial and temporal resolution comparable to these techniques. Methods:Sweating in 10 humans was stimulated on both forearms by iontophoresis of 10% acetylcholine. Silicone impressions were made and topical indicator dyes were digitally photographed every 15 seconds for 7 minutes after iontophoresis. Sweat droplets were quantified by size, location, and percent surface area. Each test was repeated eight times in each subject on alternating arms over 2 months. Another 10 subjects had silicone impressions, QDIRT, and QSART performed on the dorsum of the right foot. Results:The percent area of sweat photographically imaged correlated with silicone impressions at 5 minutes on the forearm (r = 0.92, p < 0.01) and dorsal foot (r = 0.85, p < 0.01). The number of sweat droplets assessed with QDIRT correlated with the silicone impression, although the droplet number was lower (162 ± 28 vs 341 ± 56, p < 0.01, r = 0.83, p < 0.01). The sweat response and sweat onset latency assessed by QDIRT correlated with QSART measured at the dorsum of the foot (r = 0.63, p < 0.05; r = 0.52, p < 0.05). Conclusions:The quantitative direct and indirect reflex test of sudomotor function (QDIRT) measured both the direct and the indirect sudomotor response with spatial resolution similar to that of silicone impressions, and with temporal resolution similar to that of the quantitative sudomotor axon reflex test (QSART). QDIRT provides a novel tool for the evaluation of postganglionic sudomotor function. GLOSSARYQDIRT = quantitative direct and indirect reflex test of sudomotor function; QSART = quantitative sudomotor axon reflex test; SFN = small fiber neuropathy.

T-cell response to cardiac myosin persists in the absence of an alloimmune response in recipients with chronic cardiac allograft rejection

BACKGROUND Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. METHODS The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. RESULTS AND CONCLUSIONS MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to naïve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.

Selective serotonin reuptake inhibitors to improve outcome in acute ischemic stroke: possible mechanisms and clinical evidence

Several clinical studies have indicated that selective serotonin reuptake inhibitors (SSRIs) administered in patients after acute ischemic stroke can improve clinical recovery independently of depression. Due to small sample sizes and heterogeneous study designs interpretability was limited in these studies. The mechanisms of action whereby SSRI might improve recovery from acute ischemic stroke are not fully elucidated.