Effect of oral exposure to fenitrothion and 3-methyl-4-nitrophenol on splenic cell populations and histopathological alterations in spleen in Wistar rats.

Fenitrothion (FNT) is used throughout the world as an insecticide in agriculture. To investigate the effect of FNT on the splenocytes and the underlying mechanism, FNT and its main metabolite, 3-methyl-4-nitrophenol (MNP), were administered orally to Wistar rats in daily doses of 0, 5 and 10 mg/kg, 4-5 days/week for 9 weeks. Splenocytes were harvested from control and exposed rats, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PE-CD45RA), (2) T cells (FITC-CD3), (3) T cell subsets (PE-CD4 and PerCPCD8), (4) natural killer (NK) cells (FITC-CD161a), (5) macrophages (FITC-CD11b), and (6) granulocyte (PE-granulocyte). Body weight, weight of the spleen, and histopathological alterations of spleens were also examined. The percentage of splenic CD8+ T cells and the ratio of CD8/CD4 in the group receiving 10 mg/kg FNT, and the percentages of splenic CD3+ and CD8+ T cells in the group receiving 10 mg/kg MNP were significantly decreased compared with those in the controls. FNT exposure also significantly decreased the weight of the spleen and body weight. In addition, apoptotic lymphocytes in spleen were observed in FNT-exposed rats under transmission electron microscope. However, FNT and MNP exposures did not affect splenic NK cells, B cells, macrophages, and granulocytes. The above findings indicate that FNT and MNP may selectively affect splenic T cells in rats.

Two forms of diffuse alveolar damage in the lungs of patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome is a severe disease, the treatment and pathophysiology of which are not completely established. The pathology of acute respiratory distress syndrome involves diffuse alveolar damage, which comprises severe alveolar epithelial cell damage, hyaline membrane formation, and festinate myofibroblast proliferation and fibrosis in the intra-alveolar spaces. We performed a clinicopathologic investigation of 26 autopsy cases of diffuse alveolar damage. Three cases of them were diagnosed as acute interstitial pneumonia that is idiopathic illness and resembles pathologically organizing diffuse alveolar damage. Immunohistochemical staining for types I and IV collagen, alpha-smooth muscle actin, and Ki-67 was carried out, and the sites of myofibroblast proliferation and type I collagen production were examined. All diffuse alveolar damage cases in the proliferative phase showed intra-alveolar myofibroblast proliferation. When diffuse alveolar damage was diagnosed pathologically as being due to severe infection, all 7 patients showed multiple organ dysfunction syndrome, whereas only 2 of 7 patients showed interstitial myofibroblast proliferation. When diffuse alveolar damage was attributed to tumor treatment with chemotherapy or to drug toxicity, 3 of 16 patients showed multiple organ dysfunction syndrome; 15 of 16 showed interstitial myofibroblast proliferation, 3 of 3 acute interstitial pneumonia patients did not show multiple organ dysfunction syndrome; and 3 of 3 acute interstitial pneumonia showed marked interstitial myofibroblast proliferation. These results suggest that the pathophysiologic mechanism of diffuse alveolar damage caused by severe infection is one of systemic circulation disturbance, although the mechanism underlying diffuse alveolar damage due to tumor with chemotherapy or drug toxicity appears to involve interstitial pneumonia-like lesions that are similar to acute interstitial pneumonia.

Current status and issues of C1q nephropathy.

C1q nephropathy, first proposed by Jennette and Hipp [Am J Clin Pathol 83:415-420, 1985; Am J Kidney Dis 6:103-110, 1985], was described as a distinct glomerular disease entity characterized by extensive mesangial deposition of C1q, with associated mesangial immune complexes, and the absence of any clinical and laboratory evidence of systemic lupus erythematosus. Now, 20 years since the first report, the disease entity is gradually attaining recognition, particularly in the field of pediatrics. C1q is the subcomponent of C1 in the classical pathway of complement activation. Generally, C1q deposition is caused by the activation of C1 by immunoglobulin G (IgG) and IgM; therefore, C1q nephropathy is considered as an immune complex glomerulonephritis. However, in C1q nephropathy, it remains unclear whether the deposition of C1q in the glomeruli is in response to the deposition of immunoglobulin or immune complex, or whether deposition is non-specific trapping that accompanies increased glomerular protein trafficking associated with proteinuria. Since not only the pathogenesis of C1q deposition in glomeruli but also its significance are still uncertain, it has not yet been established as an independent disease. From recent publications of the clinical and pathological characterizations, C1q nephropathy has been thought to be a subgroup of primary focal segmental glomerular sclerosis. However, many reports describe different symptoms, histopathologies, therapeutic responses and prognoses, suggesting that C1q nephropathy is not a single disease entity, but that it may be a combination of several disease groups. There are many uncertain areas requiring further investigation, though it is hoped that a detailed examination of future cases will clarify the subgroups making up C1q nephropathy and their clinicopathological characteristics, and will lead to the establishment of C1q nephropathy as an independent disease entity.

Expression of matrix metalloproteinases (MMP)-12 by myofibroblasts during alkali-burned corneal wound healing.

PURPOSE: The purpose of this study was to determine the expression of MMP-12 by myofibroblasts during the healing of alkali-burned rabbit corneas (ARC), thus implicating its role in ECM remodeling. METHODS: Rabbit corneas during alkali burn were examined for MMP-12 mRNA expression by RT-PCR. Immunohistochemistry was used to determine the presence of alpha-SMA, MMP-12 protein, and macrophages. In situ hybridization was performed to identify MMP-12 mRNA expressing cells. RESULTS: RT-PCR showed that MMP-12 mRNA was expressed in the alkali-burned corneas from one week after the injury. Immunohistochemistry showed myofibroblasts positive for MMP-12 expression. In situ hybridization revealed that MMP-12 mRNA was expressed by myofibroblasts. CONCLUSION: Our results indicate that, in alkali-burned corneas, myofibroblasts express both MMP-12 mRNA and protein. We suggest that MMP-12 may disintegrate some components of the ECM released after severe alkali burn, which may be involved in the ECM remodeling.

Ultrastructural and immunohistochemical analysis of fibrous long-spacing collagen fibrils in malignant mesothelioma.

Three cases of biphasic mesothelioma and 2 cases of sarcomatoid mesothelioma were investigated using light and electron microscopy. In 2 of the 3 cases of biphasic mesotheliomas, fibrous long-spacing (FLS) collagen fibrils were discovered with a symmetrical cross-striation of 130 nm in periodicity. However, no connection between the FLS fibrils and usual collagen fibrils were observed. Periodic acid silver methenamine stain revealed unstained bands with periods of 130 nm in FLS fibrils, whereas the usual collagen fibrils showed continuous positive staining. All 3 cases of biphasic mesotheliomas showed deposits of hyaluronic acid, whereas both cases of sarcomatoid mesotheliomas showed little hyaluronic acid. As a high concentration of hyaluronic acid induces the formation of FLS collagen fibrils in vitro, the authors propose that FLS fibrils from mesothelioma may be special structures that occur as the tropocollagens are assembled into new collagen fibrils in the presence of hyaluronic acid.

Angiotensin II receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression.

Angiotensin II receptor blockade (ARB) suppresses the progression of chronic kidney disease. However, the renoprotective effect of ARB in the active phase of glomerulonephritis (GN) has not been evaluated in detail. We examined the alteration of angiotensin II receptors' expression and the action of ARB on acute glomerular injuries in GN. Thy-1 GN was induced in rats that were divided into three groups (n=7, in each group); high dose (3 mg/kg/day) or low dose (0.3 mg/kg/day) olmesartan (Thy-1 GN+HD- or LD-ARB group), and vehicle (Thy-1 GN group). Renal function and histopathology were assessed by week 2. In the Thy-1 GN group, diffuse mesangiolysis and focal aneurysmal ballooning developed by day 3. Marked mesangial proliferation and activation progressed with glomerular epithelial injury. We confirmed that both angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R) were expressed on glomerular endothelial, mesangial, epithelial cells, and macrophages, and increased 7 days after disease induction. However, ARB treatment caused a decrease in AT1R and a further increase in AT2R expression in glomeruli. ARB prevented capillary destruction and preserved eNOS expression after diffuse mesangiolysis. Mesangial proliferation and activation was suppressed markedly with low levels of PDGF-B expression. Glomerular desmin expression, which is a marker for injured glomerular epithelial cells, was diminished significantly with retained expression of nephrin and podoplanin. Glomerular macrophage infiltration was also inhibited. Proteinuria was suppressed significantly. Furthermore, these effects of ARB showed dose dependency. These results provide insights that ARB affects individual glomerular cells and macrophages through angiotensin II receptors, with the alteration of both AT1R and AT2R expressions, and leads to inhibition of the acute destructive and proliferative glomerular lesions in GN.

Long-term inhibition of glycosphingolipid accumulation in Fabry model mice by a single systemic injection of AAV1 vector in the neonatal period.

Fabry disease is caused by the deficiency of lysosomal alpha-galactosidase A (alpha-gal A) and usually develops clinical manifestations during childhood/adolescence. Adult Fabry model mice have been successfully treated by various viral vectors. Here, in order to examine the feasibility of preventive gene therapy, we compared AAV vector-mediated gene transfer into neonatal and adult model mice. AAV serotype 1 vector (AAV1) carrying human alpha-gal A cDNA driven by the CAG promoter was intravenously injected into adult (12 weeks old) and neonatal (2 days old) Fabry model mice, and were sacrificed for detailed examination 25 weeks after vector injection. AAV1 vector preferentially transduced the liver in male adult and sustained high concentration of alpha-gal A was detected in the liver, heart and plasma. In contrast, AAV1-mediated gene expression was suppressed in similarly treated female adult mice. When the vector was systemically injected into neonates, moderate increase in plasma alpha-gal A and cardiac-specific expression of alpha-gal A were observed independently of mouse sex. The high levels of alpha-gal A activity in the heart appear to be due to the strong activity of the CAG promoter in the heart. Globotriaosylceramide (Gb3) accumulation was efficiently inhibited in the liver and heart by a single injection into both adult and neonatal animals. The biodistribution of the AAV1 vector and levels of alpha-gal A expression are markedly different between adult and neonatal mice. Neonatal injection is effective to inhibit Gb3 accumulation and therefore, might help prevent failure of major organs during adulthood.

Invagination and infolding of podocytes in glomerular basement membrane in the cases of primary membranous nephropathy.

BACKGROUND: The ultrastructural findings of membranous nephropathy (MN) are well described. Recently, podocyte infolding in the glomerular basement membrane (GBM) has been observed to be a unique ultrastructural finding formed from diffuse spherical microparticles and microtubules in the GBM. However, these alterations of glomerular epithelial cells have not been well characterized in MN. METHODS: We selected 126 renal biopsies of primary MN that were diagnosed by light microscopy and immunofluorescence. In these biopsies, we investigated the ultrastructural alterations of GBM and podocytes, especially the presence of podocyte invagination, podocyte infolding, and spherical microparticles in the GBM. RESULTS: In 98 cases (77.8%) we ultrastructurally detected occasional invagination of podocytes in the GBM within or around electron-dense or lucent deposits in mainly stage II-III of MN. In 40 cases (31.7%), we found spherical microparticles in addition to the podocyte invaginations in the GBM. In our cases, spherical microparticles were divided into three types; podocyte infolding, cell debris and virus-like particle types. Only one case displayed numerous spherical microparticles (microspheres) that were probably caused by infolding of podocytes. These microspheres, about 80 nm in diameter, were covered by unit membrane, and were accompanied by similar-sized microtubules and protrusions of podocytes. The spherical microparticles in the other cases were associated with cell debris (n = 23) or virus-like particles (n = 16) and were not connected with podocytes. CONCLUSION: Podocyte invagination associated with subepithelial deposits was a common pathological finding of primary MN, especially stage II-III of MN. The spherical microparticles in GBM in the case of MN may be associated with not only podocyte infolding but also cell debris and virus-like particles. The spherical microparticles in GBM due to diffuse podocyte infolding was considered as a new pathology finding of the GBM and may appear to be a new glomerular disease entity termed podocytic infolding glomerulopathy.

Expression of microsomal prostaglandin e synthase-1 in fibroblasts of rabbit alkali-burned corneas.

PURPOSE: Prostaglandin E2 is related to wound healing. Three different prostaglandin E synthases have been identified: microsomal prostaglandin E synthase (mPGES)-1, mPGES-2, and cytosolic prostaglandin E synthase. This study examined mPGES-1 expression in the cornea during the reparative process that occurs after an alkali burn. METHODS: mPGES-1 messenger RNA (mRNA) and protein expression levels were examined by reverse transcription-polymerase chain reaction and Western blot analysis. Localization of mPGES-1 mRNA was examined by in situ hybridization. Using immunostaining, the localization of mPGES-1, cyclooxygenase (COX)-2, and alpha-smooth muscle actin (alpha-SMA) protein was studied. RESULTS: Although mPGES-1 mRNA is expressed in normal cornea, after a corneal injury, a progressive increase of mPGES-1 mRNA occurs. In this study, 2-6 weeks after injury, mPGES-1 mRNA was detected in the stromal spindle cells. Western blot analysis also showed that mPGES-1 protein expression was observed in normal cornea, with an increase noted from 2 to 4 weeks after corneal injury. mPGES-1 immunoreactivity was negative in normal cornea; however, starting at 2 weeks after injury, positive staining of the stromal spindle cells was noted. Although COX-2 and alpha-SMA immunoreactivities were negative in the stroma of normal cornea, after injury, staining was observed in the stromal spindle cells. CONCLUSIONS: alpha-SMA-positive cells and myofibroblasts express mPGES-1 mRNA and protein, and in addition, mPGES-1 colocalized with COX-2, suggesting that myofibroblasts synthesize prostaglandin E2 and may act on and accelerate corneal wound healing.

A case of lupus nephritis with diffuse podocytic infolding into the glomerular basement membrane.

In this manuscript, we describe a case of lupus nephritis with diffuse podocytic infolding in the glomerular basement membrane (GBM). A 23-year-old woman presenting with proteinuria, leukopenia, a high value of antinuclear antibody, and positive for anti-dsDNA and anti-Sm antibodies was diagnosed with systemic lupus erythematosus. A renal biopsy was performed which showed diffuse change in the GBM and focal segmental mesangial hypercellularity under light microscopy. The GBM showed diffuse mild thickening and diffuse irregular stippling (bubble-like appearance) on staining with periodic acid-silver methenamine. However, spike formation was only occasionally seen. An immunofluorescence study was conducted which revealed fine granular deposition of IgG (2+) along the glomerular capillary walls; however, granular deposits of C1q (1+) and C3 (1+) were primarily detected in the mesangial areas. Diffuse irregular GBM thickening with dispersed distribution of microspheres and microtubules was observed using electron microscopy. In addition, these structures were chiefly detected on the epithelial side of the GBM. Since these structures seemed to connect to podocytes, we believed that the formation of these microspheres and microtubules is caused by the infolding of podocytes. This case shows a unique pathological finding, and may belong to a new glomerular disease entity characterized by podocytic infolding. After a renal biopsy, the patient received oral prednisolone 0.8 mg/kg day initially, and her complete remission status continued for 5 years.

Spatiotemporal expression of cochlin in the inner ear of rats during postnatal development.

Cochlin (encoded by COCH) constitutes 70% of non-collagenous protein in the inner ear, and the expression of cochlin is highly specific to the inner ear. Eleven missense mutation and one in-frame deletion have been reported in the COCH gene, causing hereditary progressive sensorineural hearing loss and vestibular dysfunction, DFNA9. These data imply that cochlin should bear an essential and crucial role in the inner ear function. However, the role of cochlin has not been fully clarified. We have investigated the spatiotemporal expression of cochlin in the inner ear of rats during postnatal development to better understand the functional role of cochlin. By immunohistochemistry, cochlin expression was faint in the cochlea and vestibule on the 6th day after birth (DAB6). At DAB70, strong expression of cochlin was detected in the spiral limbus and spiral ligament within the cochlea, and in the stromata of the maculae of otolithic organs and crista ampullaris within the vestibule. Immunoreactivity for cochlin increased during the postnatal development. Western blot analysis also showed an increase in the expression of cochlin isoforms. Furthermore, the dominant isoform of cochlin expressed changed from p63s to p40s between DAB24 and DAB70. These results suggest that the expression of cochlin may be related to the maturation of inner ear function, and the change in isoforms of cochlin expressed will provide important insight into the understanding of both cochlin function and formation of cochlin isoforms. This is the first to report about the spatiotemporal expression of cochlin in the developing rat inner ear.

Corneal endothelial cell damage by free radicals associated with ultrasound oscillation.

OBJECTIVE: To determine whether ultrasound oscillations in the anterior chamber cause corneal endothelial injury by free radicals. METHODS: A phacoemulsification probe was introduced into the anterior chamber of rabbits' eyes through a limbal incision, and ultrasound oscillation was performed without emulsifying the lens. Rabbits were assigned to 4 treatment groups: (1) no treatment (controls); (2) only irrigation with a salt solution; (3) ultrasound only; and (4) ultrasound oscillations with a salt solution of 0.001M ascorbic acid. The corneas were immunohistochemically examined for oxidative stress using 8-hydroxy-2-deoxyguanosine (8-OHdG), apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, and ultrastructural changes by electron microscopy. The lipid peroxide levels in the aqueous humor were also measured. RESULTS: In the ultrasound-only group, 8-OHdG-positive cells and TUNEL-positive cells were detected at 24 hours; necrotic cells were detected at 12 to 24 hours. Also, lipid peroxide levels were significantly increased at later times in the ultrasound group. Such changes were not observed in other groups. CONCLUSION: Free radicals induced by ultrasound oscillation can cause corneal endothelial damages. Clinical Relevance Clinicians should be aware that free radicals associated with ultrasound oscillation can injure the corneal endothelial cells.

Characterization of a novel congenic strain of diabetic fatty (WBN/Kob-Lepr(fa)) rat.

The WBN/Kob-Lepr(fa) rat is a new congenic strain for the fa allele of the leptin receptor gene (Lepr). Homozygous (fa/fa) WBN/Kob-Lepr(fa) rats provide a model of non-insulin-dependent diabetes with obesity. Here, we describe the characteristics of this new animal model in detail. At 7 weeks of age, both male and female obese WBN/Kob rats showed inflammatory cell infiltration of the pancreas that suggested pan-pancreatitis and an abnormal OGTT. At 3 months of age, both male and female obese WBN/Kob rats developed overt diabetes mellitus associated with severe chronic pancreatitis. In contrast, lean female WBN/Kob rats do not develop pancreatitis or diabetes. In WBN/Kob rats, this mutation might promote the onset of severe pancreatitis, leading to the rapid development of diabetes mellitus.

Apoptotic cell death and regeneration in the newborn retina after irradiation prior to bone marrow transplantation.

PURPOSE: We studied the contribution made by circulating bone marrow (BM)-derived cells to the newborn and mature retinas of BM-transplanted mice. METHODS: Newborn and adult C57BL/6J mice were administered a lethal dose of total-body irradiation, after which pathologic changes to the retinas were periodically assessed. In addition, mice received BM cells from 8-week-old green fluorescent protein (GFP) transgenic mice, and the subsequent differentiation of GFP+ cells was studied. RESULTS: Within 5 hr after irradiation of newborn mice, retinal cells began to die due to apoptosis. By contrast, irradiation of adult mice elicited no histologic changes in the retina. BM cells generally did not differentiate in adult mice, but numerous GFP+ BM cells were integrated into the retinal tissue of newborn mice, where they expressed various cell type-specific markers. Finally, examination of whole retina mounts showed that GFP+ cells also contributed to retinal vascularization. CONCLUSIONS: Our findings underscore the importance of careful evaluation of the biological effects of irradiation in models making use of BM transplantation.

Systemic cancer gene therapy using adeno-associated virus type 1 vector expressing MDA-7/IL24.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.

Role of matrix metalloproteinases and their tissue inhibitor of metalloproteinases in myxomatous change of cardiac floppy valves.

To clarify the underlying cause of myxomatous changes in cardiac floppy valves, the expression of the matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) was investigated in cardiac valves. Valves were obtained from nine patients with floppy valves, from 13 patients with other valvular disease types, and from four patients with normal valves. Immunohistochemical analyses for MMP-2, MMP-9, TIMP-1, and TIMP-2, and gelatin zymography for MMP-2 and MMP-9 were performed. Compared with the spongiosa of normal valves, the myxomatous area of floppy valves had stronger immunohistochemical reaction to MMP-2 and MMP-9, and weaker reaction to TIMP-2. Activated MMP-2 and MMP-9 were detected in eight out of nine cases of floppy valves. Activated MMP-2 was detected at low levels in two cases of normal valves showing mild expansion of the spongiosa without macroscopic floppiness. The ratio of active/total MMP-2 and MMP-9 increased in floppy valves compared with normal valves. These results suggest that the imbalance between MMP and TIMP and the increased activity of MMP-2 and MMP-9 may correlate with myxomatous changes observed in floppy valves. Valves with a slight myxomatous change and activated MMP-2 may develop into floppy valves with increases in the activity of MMP.

Immunohistochemical observation of amniotic membrane patching on a corneal alkali burn in vivo.

PURPOSE: To investigate by immunohistochemical observation the effects of amniotic membrane (AM) patching on myofibroblastic differentiation and matrix metalloproteinase (MMP) expression in the corneal stroma after an alkali burn in vivo. METHODS: A corneal alkali burn was made by placing a circular piece of filter paper containing 1 N NaOH on the central cornea of rabbits. Burning was done unilaterally in each rabbit. Immediately after the wounding, in the AM group, AM was sutured onto the cornea and removed on day 1. Rabbits with no AM patching were controls. On day 14, corneas were excised and immunohistochemical observation was carried out using antibodies against alpha-smooth muscle actin (alpha-SMA), vimentin, MMP-1, MMP-2, MMP-9, and membrane-type1 (MT1)-MMP. Observation after Masson trichrome staining was also performed. RESULTS: In the AM group, alpha-SMA positive cells were noticeably fewer, and MMP-2, MMP-9, and MT1-MMP expression was clearly inhibited. Also, collagen fibers were more regularly arranged than in control eyes. The more proximate the cells were to the epithelial side, the fewer alpha-SMA-positive cells were observed in the AM group. CONCLUSIONS: AM patching suppressed myofibroblastic differentiation and MMP expression in the stroma after an alkali burn. An inhibition gradient suggests that AM may release unknown soluble factors possessing some antiscarring capability.

Immunogenicity and antigenicity of allogeneic amniotic epithelial transplants grafted to the cornea, conjunctiva, and anterior chamber.

PURPOSE: To determine the immunogenic characterization of amniotic epithelium (AE), by examining the fate of allogeneic AE grafts heterotopically transplanted in the eye. METHODS: Intact AE from enhanced green fluorescence protein (EGFP) transgenic mice (C57BL/6 background) and wild-type C57BL/6 mice were transplanted onto cornea or conjunctiva, or inserted into the anterior chamber (AC) of normal BALB/c mice, C57BL/6 mice, or BALB/c mice presensitized to donor antigens. For repeated AE transplantation experiments, AE was grafted in the other eye 7 days after the first grafting. Graft fate was assessed clinically and histologically at selected intervals after grafting. Infiltrating inflammatory cells were examined immunohistochemically. Sensitization to alloantigens by AE was assessed by the delayed hypersensitivity (DH) response. RESULTS: In normal recipients, GFP+ cells were absent in EGFP donor-derived AE grafts by day 21 on cornea and by day 7 on conjunctiva. AE grafts implanted in the AC survived for >8 weeks. In presensitized recipients and recipients that underwent repeated AE implantation, graft survival was markedly shorter than in normal recipients. DH was induced at 2 weeks, but failed to be induced at 4 weeks after grafting on cornea or at 8 weeks after grafting on conjunctiva and in the AC of normal recipients. CONCLUSIONS: Fresh allogeneic AE expressed immunogenicity when placed on the ocular surface, although no memory of allospecific DH was acquired. Allogeneic AE is clearly vulnerable to immune rejection in specifically sensitized recipients.

Expression of cochlin in the vestibular organ of rats.

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Cochlin constitutes 70% of the inner ear protein and cochlin isoforms can be classified into three subgroups, p63s, p44s and p40s. Symptoms of some DFNA9 patients are consistent with those of Ménière's disease. Here, we report the expression of cochlin in the vestibular organ of rats using isoform-specific antibodies that recognize all three isoforms. Cochlin is highly expressed in the stromata of the maculae of otolithic organs and cristae of semicircular canals, and in the channels in the bony labyrinth that transmit the dendritic innervation to the cristae and maculae. Cochlin cannot be detected in the sensory cells, dark cells, nor in the acellular structures, otolithic membrane or in the cupula. These findings support the theory that deposition of acidophilic substance in the inner ear caused by mutation of cochlin can induce a secondary retrograde dendritic degeneration of the vestibular nerves.