Outcomes of patients receiving a massive transfusion for major trauma.

BACKGROUND: The benefits of high transfusion ratios (plasma to red blood cells and platelets to red blood cells) on survival in injured patients who receive massive transfusions remain uncertain. This study aimed to assess the association between transfusion ratios and adverse events and survival in patients undergoing massive transfusion for major trauma. METHODS: A retrospective observational study was conducted on patients who had major trauma using a Japanese national administrative database. The associations between transfusion ratios and outcomes (in-hospital mortality and incidence of adverse events) were analysed using a non-linear logistic generalized additive model (GAM). In a logistic generalized estimating equation model, adjusted for patient and hospital-level confounders, transfusion ratios were included as continuous or categorical variables (low, transfusion ratio 0·75 or less; intermediate, over 0·75 to 1·25; high, over 1·25). RESULTS: Some 1777 patients were included in the analysis, of whom 602 died in hospital. GAM plots of the transfusion ratios for in-hospital mortality demonstrated a downward convex unimodal curve. In-hospital mortality was similar with increasing transfusion ratios for plasma (adjusted odds ratio (OR) 1·13, 95 per cent c.i. 0·82 to 1·55; P = 0·446) and platelets (adjusted OR 0·84, 0·66 to 1·08; P = 0·171). Both plasma to red blood cell ratio (adjusted OR 1·77, 1·32 to 2·37; P < 0·001) and platelet to red blood cell ratio (adjusted OR 1·71, 1·35 to 2·15; P < 0·001) were significantly associated with a higher incidence of adverse events. No significant differences in in-hospital mortality were observed between the three transfusion categories (low, medium and high). CONCLUSION: In this study, transfusion strategies with high plasma to red blood cell and platelet to red blood cell ratios did not have survival benefits, but were associated with an increase in adverse events.

Effectiveness of early administration of tranexamic acid in patients with severe trauma.

BACKGROUND: A reduction in mortality with the early use of tranexamic acid has been demonstrated in severely injured patients who are bleeding. However, the modest treatment effect with no reduction in blood transfusion has raised concerns. The aim of the present study was to estimate the effectiveness of regular use of tranexamic acid in severely injured patients. METHODS: This multicentre observational study used retrospectively collected data from consecutive injured patients (Injury Severity Score at least 16) treated in 15 Japanese academic institutions in 2012. A propensity score-matched analysis compared patients who did or did not receive tranexamic acid administration within 3 h of injury. Study outcomes included 28-day all-cause and cause-specific mortality, and need for blood transfusion. RESULTS: Of 796 eligible subjects, 281 were treated with tranexamic acid. Propensity score matching selected a total of 500 matched subjects (250 in each group). Tranexamic acid administration was associated with lower 28-day mortality (10·0 versus 18·4 per cent; difference -8·4 (95 per cent c.i. -14·5 to -2·3) per cent) and lower 28-day mortality from primary brain injury (6·0 versus 13·2 per cent; difference -7·2 (-12·3 to -2·1) per cent). However, there was no significant difference between groups in the need for blood transfusion (33·2 versus 34·8 per cent; difference -1·6 (-9·9 to 6·7) per cent). CONCLUSION: Early tranexamic acid use was associated with reduced mortality in severely injured patients, in particular those with a primary brain injury.

Efficacy and safety of ustekinumab in Japanese patients with severe atopic dermatitis: a randomized, double-blind, placebo-controlled, phase II study.

BACKGROUND: Ustekinumab, a fully human monoclonal antibody against interleukin-12/23, may potentially be effective for severe atopic dermatitis (AD) treatment. OBJECTIVES: To evaluate efficacy and safety of ustekinumab 45 mg and 90 mg in patients with severe AD. METHODS: In this randomized, placebo-controlled, phase II study, Japanese patients (aged 20-65 years) with severe or very severe AD entered a 12-week double-blind treatment period during which they received (1 : 1 : 1) ustekinumab 45 mg, 90 mg or placebo subcutaneous injections at weeks 0 and 4, with follow-up until week 24. The primary efficacy end point was percentage change from baseline in Eczema Area and Severity Index (EASI) score at week 12. Major secondary efficacy end points included the proportion of patients achieving EASI 50, EASI 75, Investigator's Global Assessment score 0-1, change from baseline Atopic Dermatitis Itch Scale and Dermatology Life Quality Index. RESULTS: A total of 79 patients were randomized [ustekinumab 45 mg (n = 24), 90 mg (n = 28), placebo (n = 27)]. Ustekinumab treatment showed nonsignificant improvement in least square mean change from baseline EASI score at week 12 [45 mg: -38·2%, 95% confidence interval (CI) -21·02-19·51; P < 0·94 and 90 mg: -39·8%, 95% CI -21·84-17·14; P < 0·81] vs. placebo (-37·5%). A nonsignificant improvement in major secondary efficacy end points was observed in both ustekinumab groups vs. placebo. The most common treatment-emergent adverse events were nasopharyngitis and worsened AD (higher in placebo vs. ustekinumab groups). CONCLUSIONS: Ustekinumab 45 mg and 90 mg did not demonstrate meaningful efficacy in Japanese patients with severe AD. The treatment was generally well tolerated.

Association between BIA-derived Phase Angle and Sarcopenia and Improvement in Activities of Daily Living and Dysphagia in Patients undergoing Post-Stroke Rehabilitation.

OBJECTIVES: To investigate the predictive value of the BIA-derived phase angle with respect to the functional prognosis and baseline sarcopenia in patients undergoing post-stroke rehabilitation. DESIGN: Retrospective cohort study. SETTING AND PARTICIPANTS: Overall, 577 Japanese patients admitted to a post-acute care hospital from 2016 to 2020 were recruited. MEASUREMENTS: Body composition analysis, which included BIA-derived phase angle and skeletal muscle mass, was performed using bioelectrical impedance analysis (BIA). Study outcomes included physical function assessed using the Functional Independence Measure (FIM-motor) and the level of dysphagia assessed using the Food Intake LEVEL Scale (FILS). Sarcopenia was defined as the loss of skeletal muscle mass and decreased muscle strength. Receiver operating characteristic curves were used to calculate the optimal cutoff value of BIA-derived phase angle to diagnose sarcopenia. Multivariate analyses were used to determine whether the BIA-derived phase angle at admission was associated with outcomes at discharge and baseline sarcopenia. RESULTS: After enrollment, 499 patients (mean age: 74.0 ± 13.1 years; 52.0% men) were examined. The median FIM-motor and FILS scores at admission were 47 (20-69) and 8 (7-10), respectively. Sarcopenia was observed in 43.2% of patients. After adjusting for potential confounders, BIA-derived phase angle was positively associated with FIM-motor scores at discharge (ß = 0.134, P < 0.001), FIM-motor score gain (ß = 2.504, P < 0.001), and FILS scores at discharge (ß = 0.120, P = 0.039). BIA-derived phase angle was negatively associated with the sarcopenia diagnosis at baseline (odds ratio = -0.409, P < 0.001); its cutoff value was 4.76° (sensitivity 0.800, specificity 0.790, P < 0.001) for sarcopenia diagnosis in men and 4.11° (sensitivity 0.735, specificity 0.829, P < 0.001) in women. CONCLUSION: BIA-derived phase angle was positively associated with the recovery of physical function and dysphagia level and negatively associated with baseline sarcopenia in patients undergoing post-stroke rehabilitation. The BIA-derived phase angle cutoff for sarcopenia diagnosis was 4.76° for men and 4.11° for women.

Oral Management in Rehabilitation Medicine: Oral Frailty, Oral Sarcopenia, and Hospital-Associated Oral Problems.

Oral health is a crucial but often neglected aspect of rehabilitation medicine. Approximately 71% of hospitalized rehabilitation patients and 91% of hospitalized acute care patients have impaired oral health. Poor oral condition in hospitalized patients can be attributed to factors such as age, physical dependency, cognitive decline, malnutrition, low skeletal muscle mass and strength, and multimorbidity. Another major factor is a lack of knowledge and interest in oral problems among health care workers. Recently, new concepts have been proposed, such as oral frailty, oral sarcopenia, and hospital-associated oral problems. Oral frailty, the accumulation of a slightly poor status of oral conditions and function, strongly predicts physical frailty, dysphagia, malnutrition, need for long-term care, and mortality in community-dwelling older adults. Oral sarcopenia refers to sarcopenia associated with oral conditions and function, although its definition has not yet been fully discussed. Hospital-associated oral problems are caused by disease, disease treatment, surgery, endotracheal intubation, poor self-care abilities, lack of care by medical staff, drugs, and iatrogenic factors during hospitalization. Furthermore, oral problems have negative impacts on rehabilitation outcomes, which include functional recovery, length of hospital stay, discharge home, and in-hospital mortality. Oral health management provided by dental hygienists improves not only oral status and function, swallowing function, and nutritional status but also activities of daily living, discharge home, and in-hospital mortality in post-acute rehabilitation. Oral rehabilitation, promotion, education, and medical-dental collaboration can be effective interventions for oral problems and therefore are necessary to improve rehabilitation outcomes.

Expansion of radiofrequency ablation volume by saturated NaCl saline injection in the area of vaporization.

BACKGROUND: Vaporization around the radiofrequency (RF) electrode after RF application (RFA) limits the RF ablation area. PURPOSE: To determine whether saturated saline injected into the area of vaporization after initial RFA extends ablation area after further RFA. MATERIAL AND METHODS: RFA was performed in 18 ex vivo porcine livers and four in vivo rabbit erector spinae muscles. An RF electrode was used to ablate an area with 40W of parallel current for 15 min. The ablation margin was determined using a thermocouple, and the radius of the ablated area was measured. After RF electrode removal, saturated saline was infused through a percutaneous ethanol injection needle into the site of the original RFA in 11 liver samples and two erector spinae muscles. Three minutes later, RFA was resumed for 15 min. The remaining seven control liver samples and two spinae muscles received RFA without saline injection. The radius of the final ablated area was then measured. RESULTS: In the ex vivo study, injection of saturated saline significantly decreased tissue impedance (87.7+/-9.4 to 51.1+/-9.7 Omega, P<0.0001), and increased the mean radius of the ablated area (15.9+/-3.0 to 25.0+/-3.6 mm, P<0.0001). These significant changes were not observed without injection of saturated saline. Similar trends were found in the in vivo study. CONCLUSION: Injection of saturated saline into the area of vaporization around the RF electrode, followed by additional RFA, caused concentric expansion of the final ablation area, facilitating more efficient tumor ablation.

Oral administration of dihomo-gamma-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.

Disorders of the metabolism of essential fatty acids (EFAs) are related to atopic dermatitis (AD). Concentrations of dihomo-gamma-linolenic acid (DGLA), an EFA, in the serum of AD patients are lower than those in healthy volunteers. Recently we developed a fermented DGLA oil, and examined whether oral administration of DGLA prevents development of dermatitis in NC/Nga mice, which spontaneously develop human AD-like skin lesions. NC/Nga mice were fed a diet either containing or not containing DGLA for 8 weeks under in air-uncontrolled conventional circumstances. Clinical skin severity scores were significantly lower in mice fed DGLA than in mice not fed it. Scratching behavior and plasma total IgE levels were also reduced in the DGLA group, in association with histological improvement. DGLA suppressed clinical severity of skin lesions dose-dependently, with an increase in DGLA contents in phospholipids of skin, spleen, and plasma. Discontinuation of DGLA administration resulted in the onset of dermatitis and a decrease in DGLA contents in skin, spleen, and plasma. These findings indicate that oral administration of DGLA effectively prevents the development of AD in NC/Nga mice, and that DGLA in phospholipids is a compound of key importance in the development and prevention of dermatitis.

Methylnitrosourea-induced tumorigenesis in MGMT gene knockout mice.

Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase [Tsuzuki et al., Carcinogenesis (Lond.), 17: 1215-1220, 1996]. These MGMT-/- mice were most sensitive to the alkylating carcinogen, methylnitrosourea; when varied doses of methylnitrosourea were administered to 6-week-old mice and survivals at the 30th day were determined, LD50s of MGMT-/- and MGMT+/+ mice were 20 and 240 mg/kg of body weight, respectively. MGMT+/- mice were as resistant as MGMT+/+ mice, but some difference in survival time was noted when the two genotypes of mice were exposed to a relatively high dose of methylnitrosourea. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT-/- mice exposed to methylnitrosourea at a dose of 2.5 mg/kg of body weight. In case of exposure to the same dose of drug, no or few tumors occurred in the MGMT+/+ and MGMT+/- mice. It appears that the DNA repair methyltransferase protein protected these mice from methylnitrosourea-induced tumorigenesis.

Stimulation of the labellar sugar receptor of the fleshfly by mono- and disaccharides.

Responses of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were studied over a wide range of concentrations of several sugars (sucrose, maltose, glucose, fructose, and mannose) in single solutions and in mixtures. The results suggest (a) that the receptor sites are not completely differentiated for glucose and for fructose combination, (b) that the receptor site is composed of two subunits. Such suggestions are based on the classical model, where the response is proportional to the number of the sites, two subunits of each site being simultaneously occupied with one molecule of disaccharides or two molecules of monosaccharides. It is shown, however, that an allosteric model gives a somewhat better interpretation of the experimental results.

Stimulation of the labellar sugar receptor of the fleshfly by mono- and disaccharides.

Responses of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were studied over a wide range of concentrations of several sugars (sucrose, maltose, glucose, fructose, and mannose) in single solutions and in mixtures. The results suggest (a) that the receptor sites are not completely differentiated for glucose and for fructose combination, (b) that the receptor site is composed of two subunits. Such suggestions are based on the classical model, where the response is proportional to the number of the sites, two subunits of each site being simultaneously occupied with one molecule of disaccharides or two molecules of monosaccharides. It is shown, however, that an allosteric model gives a somewhat better interpretation of the experimental results.

The effects of pH on the labellar sugar receptor of the fleshfly.

Reproducible results describing the effects of pH on the response of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were obtained. The response to sucrose was independent over a wide range of pH (3.0 to 10.0 for sucrose stimulation), but was inhibited fairly sharply on both sides of this range. Similar results were obtained for monosaccharide stimulation. The receptor was excited on stimulation by water above pH 12.0. The effects of high pH, both inhibitory and excitatory, were affected by the presence of salts. In the presence of 0.5 molar NaCl, for example, the pH-inhibition curve was shifted toward lower pH's by about one pH unit. The effects of low pH, on the other hand, were not affected by salts. Following Dixon's theory, it was concluded that at least five ionizable groups (loosing positive charges above pH 10.5) were located at the receptor site.

The effects of amino acids on the labellar hair chemosensory cells of the fly.

The effects of amino acids on the labellar hair chemosensory cells were examined with two kinds of flies (the fleshfly, Boettcherisca peregrina, and the blowfly, Phormia regina). As a result of this examination, the effects of amino acids were divided into four main classes. Amino acids in class 1 did not stimulate any chemoreceptor cell. Amino acids in class 2 inhibited nonspecifically the discharges from three kinds of chemosensory cells. Amino acids in class 3 stimulated the salt receptor cell. Amino acids in class 4 stimulated the sugar receptor cell. A possibility that a fourth neuron in the labellar hair chemosensory cell might be a protein or an amino acid receptor cell was eliminated.

Alfacalcidol inhibits bone resorption and stimulates formation in an ovariectomized rat model of osteoporosis: distinct actions from estrogen.

Although alfacalcidol has been widely used for the treatment of osteoporosis in certain countries, its mechanism of action in bone, especially in the vitamin D-replete state, remains unclear. Here we provide histomorphometric as well as biochemical evidence that alfacalcidol suppresses osteoclastic bone resorption in an ovariectomized rat model of osteoporosis. Furthermore, when compared with 17beta-estradiol, a representative antiresorptive drug, it is evident that alfacalcidol causes a dose-dependent suppression of bone resorption, and yet maintains or even stimulates bone formation, as reflected in increases in serum osteocalcin levels and bone formation rate at both trabecular and cortical sites. 17beta-Estradiol, which suppresses bone resorption to the same extent as alfacalcidol, causes a parallel reduction in the biochemical and histomorphometric markers of bone formation. As a final outcome, treatment with alfacalcidol increases bone mineral density and improves mechanical strength more effectively than 17beta-estradiol, with a more pronounced difference in cortical bone. We conclude that estrogens depress bone turnover primarily by suppressing bone resorption and, as a consequence, bone formation as well, whereas alfacalcidol "supercouples" these processes, in that it suppresses bone resorption while maintaining or stimulating bone formation.

Humoral mediator-dependent activation of the Sarcophaga lectin gene.

Sarcophaga lectin is a defence protein synthesized by the fat body and secreted into the hemolymph in response to injury of the body of third instar larvae of Sarcophaga peregrina (flesh-fly). In this paper, we demonstrate that the stimulus of body injury is first transmitted to a certain tissue present in the anterior part of the body, and from there a mediator molecule that interacts directly with fat body cells is secreted into the hemolymph. On interaction with this mediator molecule, the fat body begins to synthesize mRNA for Sarcophaga lectin.

The calcitonin-like sequence of the beta CGRP gene.

We have identified a region within the beta CGRP gene which has the potential to encode a novel calcitonin-like peptide. The gene is located on the short arm of chromosome 11 (11p 12-14.2) and we suggest that it resulted from a local duplication of the alpha gene. We have been unable to detect the corresponding mRNA in a variety of tissues which express alpha-calcitonin. It is not clear whether this sequence can be expressed in man.

Contribution of trabecular and cortical components to the mechanical properties of bone and their regulating parameters.

To evaluate the mechanical contributions of the spongiosa and cortex to the whole rat vertebra, we developed a finite element analysis (FEA) system linked to three-dimensional data from microcomputed tomography (micro-CT). Twenty-eight fifth lumbar vertebrae (L-5) were obtained from 10-month-old female rats, comprised of ovariectomized (ovx, n = 6), sham operated (n = 7), and alfacalcidol-treated after ovx (0.1 microg/kg [n = 8] and 0.2 microg/kg [n = 7]) groups. The trabecular microstructure of L-5 was measured by micro-CT. Yield strength at the tissue level (YS), defined as the value at which 0.034% of all elements reached yield stress, was calculated by the FEA. Then, the ultimate compressive load of each specimen was measured by mechanical testing. The YS of the whole bone (YSw) showed a significant correlation with ultimate load (r = 0.91, p < 0.0001). The YS values of the isolated spongiosa (YSs) and cortex (YSc) were calculated in models with varying amounts of trabecular or cortical bone mass. The mechanical contribution of the spongiosa showed a nonlinear relationship with bone mass, and ovx reduced the mean mechanical contribution of the spongiosa to the whole bone by 13% in comparison to the sham group. YSs had a strong relationship with trabecular microstructure, especially with trabecular bone pattern factor (TBPf) and structure model index (SMI), and YSc had a strong relationship with cortical bone volume. The structural parameters most strongly related to YSw were BV/TV and TBPf. Our micro-FEA system was validated to assess the mechanical properties of bone, including the individual properties of the spongiosa and cortex, in the osteoporotic rat model. We found that the mechanical property of each component had a significant relationship with the respective bone mass, volume, or structure. Although trabecular microstructure has a significant relationship with bone strength, in ovx bone with deteriorated trabecular microstructure, the strength depended mainly on the cortical component.

Effect of trabecular bone contour on ultimate strength of lumbar vertebra after bilateral ovariectomy in rats.

To test the hypothesis that the effect of trabecular microarchitecture on bone strength varies with the duration of estrogen loss, we evaluated the relationship between three-dimensional (3D) parameters for trabecular microarchitecture and bone minerals with the compressive load of the lumbar vertebra in rats. Female Sprague-Dawley rats (n = 190) were divided into 19 groups. Ten rats were killed at day 0. Half of the remaining rats underwent bilateral ovariectomy (ovx), and the others were subjected to sham surgery. Ten rats from each group were killed at 3, 7, 11, 14, 28, 42, 56, 70, and 84 days postsurgery. Urinary deoxypyridinoline and serum osteocalcin increased significantly in the ovx group from days 28 and 11, respectively, compared with the sham group. Bone mineral content (BMC) and bone mineral density (BMD) of the fifth lumbar body diminished from days 42 and 84, respectively, compared with the sham group. In ovx rats, trabecular bone volume (BV/TV), measured using 3D images of microcomputed tomography, diminished from day 28 compared with both baseline control and sham. The trabecular bone pattern factor (TBPf) and structure model index (SMI) increased from day 28 in the ovx group compared with both baseline control and sham. Ultimate compression loads diminished at day 28 compared with baseline control and decreased progressively thereafter. Neither of these parameters changed in the sham group during the same period. Within 4 weeks post-ovx, TBPf, SMI, and BV/TV correlated with load (p < 0.01). BMC and BMD correlated with load from 6 weeks post-ovx (p < 0.01). Stepwise regression analysis showed that TBPf was the most significant determinant of load within 4 weeks post-ovx (coefficient of determination [R(2)] = 0.669; p < 0.01). SMI correlated with TBPf (R(2) = 0.968; p < 0.01). Moreover, R(2) for ultimate load indicated higher values of 0.975 with TBPf and SMI. However, BMC was the most significant determinant of load from 6 weeks post-ovx (R(2) = 0.511; p < 0.01), as it was in the sham group. These data suggest that changes in trabecular bone contour with increased bone turnover are critical for reducing lumbar bone strength during the early post-ovx period in rats.

Identification of the cornea-specific keratin 12 promoter by in vivo particle-mediated gene transfer.

PURPOSE: Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, it is necessary to identify and characterize the promoter of Krt1.12 gene. METHODS: The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. Krt1.12 promoter-beta-gal DNA constructs were prepared and used in vivo to transfect rabbit corneas, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). In vitro, the DNA constructs were transfected into cultured T-antigen-transformed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-1080 fibroblasts with lipofectamine. The promoter activity was assessed by measuring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o-nitrophenyl beta-D-galactopyranoside. RESULTS: There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 gene. None of promoter constricts can be expressed by HT-1080 cells. Cotransfection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax-6 elements results in a fourfold increase of beta-gal activities in RCE-T cells but not HT-1080 fibroblasts. The data of in vivo transfection in the rabbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells. CONCLUSIONS: The particle-mediated gene transfer is a suitable technique for in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DNA fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific regulatory cis-DNA elements. Pax-6 is a positive transcription factor essential for keratin 12 expression.

Keratin 12-deficient mice have fragile corneal epithelia.

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.

Behavioral sensitization to beta-phenylethylamine (PEA): enduring modifications of specific dopaminergic neuron systems in the rat.

Repeated daily administration of an endogenous trace amine, beta-phenylethylamine (PEA), produces behavioral sensitization such that the intensity of PEA-induced stereotyped behaviors in rats increases gradually during the treatment, and a challenge injection with PEA reinstates the enhanced stereotypy even long after withdrawal. In the present study, we examined the neurochemical changes in the central dopaminergic neurons systems in the rat for 7 drug-free days after repeated treatment with PEA (50 mg/kg, IP day for 14 or 28 days). During withdrawal, a decrease in steady-state levels of tissue dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), was found in the mesolimbic DA nerve terminal areas of the rat brain receiving repeated PEA treatment. Fifteen minutes after challenge administration of PEA at varying doses from 6.3 to 75 mg/kg, the rats with repeated PEA treatment required smaller doses of PEA challenge than the rats with acute PEA treatment in order to obtain a significant decrease in striatal DOPAC content compared to the saline control in each treatment group. These results imply that the behavioral sensitization to PEA is accompanied by enduring modifications of the specific dopaminergic neuron systems in the rat brain. This suggestion was strongly supported by the results of the study using in vivo intracerebral dialysis, which indicated that 25 mg/kg PEA challenge elicited a remarkable increase in the extracellular DA concentrations in striatal perfusates collected from the PEA-pretreated rats, in accordance with the intensity of stereotyped behaviors. These findings argue that the hyper-responsiveness to PEA of the striatal dopaminergic neuron systems persists long after withdrawal from repeated treatment with PEA.