Hoxa-10 and Cyclin D3 Overexpression in the Decidual Reaction in a Superovulation Protocol in Young Adult C57BL/6J Mice.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

Cisplatin as a Viable and Secure Alternative to Carmustine in BEAM-Based Conditioning for Autologous Hematopoietic Stem Cell Transplantation in Patients with Lymphoma.

BACKGROUND: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) is a standard treatment for relapsed/refractory lymphoma patients. Yet, the widespread use of BEAM is hindered by carmustine accessibility. This study evaluates the efficacy and safety of PEAM (Cisplatin, Etoposide, Cytarabine, and Melphalan) versus BEAM in auto-HSCT for Hodgkin (HL) and non-Hodgkin lymphoma (NHL) patients. METHODS: We conducted a retrospective single-center study of adult lymphoma patients who received PEAM or BEAM pretransplant conditioning between January 2004 to December 2022, comparing efficacy and safety outcomes. RESULTS: Among 143 patients (median age of 33 years, 58% males), 55 had HL, and 88 had NHL. The overall response rate (ORR) was 86.7% for PEAM and 72.3% for BEAM, and the relapse rate (RR) was lower for PEAM than BEAM (22.9% vs 45.6%). Median time to relapse (TTR) and overall survival (OS) were not reached for either group. PEAM exhibited a shorter time to both neutrophil (NE) and platelet (PE) engraftment compared to BEAM (10 vs 12 days), with a more tolerable gastrointestinal (GI) toxicity profile. CONCLUSIONS: Both BEAM and PEAM showed similar outcomes, demonstrating comparable efficacy in terms of ORR, TTR, and OS for both HL and NHL patients. However, PEAM-conditioning was associated with a shorter time to engraftment and fewer GI adverse events.

[The etiology and management of gout]. / Ätiologie und Management der Gicht.

Gout is an inflammatory arthritis caused by monosodium urate (MSU) crystal deposits in and around the joint. The formation of urinary calculi can also occur in gout, but are less common than arthritis. Gout usually presents with recurrent episodes of joint inflammation, which over time lead to tophus formation and joint destruction. In the last decade, significant advances have been made regarding not only the epidemiology and genetics of gout and hyperuricemia but also the mechanisms of inflammation and treatment of gout. In addition, knowledge concerning the key role of interleukin 1 (IL-1) has provided new therapeutic perspectives. However, the current management of gout is often suboptimal, with many Patienten either not receiving adequate treatment or being unable to tolerate existing treatments. New therapeutic agents provide interesting new options for Patienten with difficult-to-treat gouty arthritis.The English full-text version of this is available at SpringerLink (under "Supplemental").

[Outbreaks of acute gastroenteritis in Antofagasta Region, Chile 2010]. / Brote de gastroenteritis aguda en la Región de Antofagasta,Chile: 2010.

Outbreaks of acute gastroenteritis are a public health problem. Norovirus is known as the most common cause (50%). In Chile, immediate notification allows surveillance of these events. We describe an acute gastroenteritis outbreak that occurred in Antofagasta region, between March and April 2010. An observational study was conducted to perform the outbreak investigation. Local residents who met case definition were included. Stool samples, epidemiological surveys and environmental samples were requested. The outbreak began approximately on March 8, 2010 and lasted until April 28 with 31,036 reported cases (rate 54 per 1000 inhabitants). The most affected age group was between 25 and 44 years, and diarrhea was the main symptom (97% of cases). We determined the presence of norovirus genogroup II in clinical and environmental samples. This outbreak was caused by consumption of raw vegetables from La Chimba, which were watered and contaminated with treated sewage containing low concentration of free residual chlorine. Subsequently, the outbreak spread from person to person in a poor sanitary environment.

[Antimicrobial susceptibility, capsular serotypes and clonal relationship of invasive streptococcus pneumoniae isolates in adult population of the Bio-Bio region, Chile. 2005-2006]. / Susceptibilidad antimicrobiana, serotipos capsulares y relación clonal entre cepas invasoras de streptococcus pneumoniae aisladas de pacientes adultos de la Región del Bío-Bío, Chile. Periodo 2005-2006.

Streptococcus pneumoniae infections constitute a public health problem. In our country there is scarce information regarding isolates from bacteraemic episodes in adult population. The antibiotic susceptibility, serotypes and clonal relationship of 56 isolates of S. pneumoniae from adult patients with bacteraemic infections in Concepcion-Talcahuano, Bio-Bio Region, Chile, were studied. Resistance to tetracycline (21.4%), trimethoprim/ sulfamethoxazole (18%), erythromycin (18%), chloramphenicol (7%) and 1 penicillin resistant isolate from a meningeal focus (2%) was found. Also, all the isolates were susceptible to cefotaxime, levofloxacin, moxifloxacin and vancomycin. A wide variety of capsular serotypes was demonstrated, with predominance of serotypes 1, 5, 23F, 7F and 3. The macrorestriction analysis by pulse field electrophoresis revealed 31 electrophoretic patterns and 12 clonal groups, discarding a predominant clone. According to the results, at least, 80% of the S. pneumoniae serotypes isolated from bacteraemic adult patients are included in the available commercial vaccine.

Urban birds: An important source of antimicrobial resistant Salmonella strains in Central Spain.

Antimicrobial resistance (AMR) is one of the most important threats of the 21 st century. Wild birds have been described as reservoirs of AMR in different bacterial species, such as Salmonella spp. Privation of food, climate change and overpopulation have forced many wild species to modify their feeding habits, attending urban areas. In this context, the aim of this study was to study Salmonella presence, as well as related AMR in urban birds that inhabit the city and its surroundings. A total of 300 urban birds were sampled for Salmonella detection according to the ISO 6579-1:2017 (Annex D) recommendations, and serotyping was carried out according to the White-Kauffman-Le Minor scheme. Antimicrobial susceptibility was tested following 2013/652/EU Decision guides. Wild birds analysed were positive for Salmonella in 12.3 % of cases, with white storks fed in landfills as the most Salmonella prevalent species (p < 0.05). The most common serovars isolated were zoonotic (S. Enteritidis, S. Typhimurium and S. Typhimurium monophasic variant). From Salmonella isolated strains, 40.5 % were resistant to the most prevalent AMRs found in urban birds were ciprofloxacin (36.4 %), nalidixic acid (36.4 %) and colistin (27.3 %). The scientific community, public administration and population in general should work together to control antimicrobial administration and drug waste management in order to decrease the development and spread of AMR.

A systematic review of tools used to screen and assess for externalising behaviour symptoms in low and middle income settings.

BACKGROUND: Mental health issues, often manifested as behavioural difficulties, in children are estimated to be high in low and middle-income countries (LMIC) settings. There is a paucity of definitive data due to a lack of well-validated tools to use across settings. This review aims to provide evidence on what tools are used and which have been adapted and validated in LMIC settings. METHODS: We performed a systematic review to identify tools used to assess or screen externalising behaviour problems in children and adolescents in LMIC and assess their cultural adaptations. We searched for studies measuring externalising behaviour in children from 0 to 19 years published up to September 2018. Articles were assessed to identify tools used and analysed using the Ecological Validity Framework. RESULTS: We identified 82 articles from over 50 LMICs who had studied externalising behaviour in children. Twenty-seven tools were identified, with a predominance of studies using tools from the USA and Europe. Most studies did not describe an adaptation and evaluation process, with only one study following recommended criteria. New tools were identified which both screen and assess externalising behaviour which have not yet been utilised across settings. CONCLUSIONS: Although tools from the USA and Europe are often utilised to screen and assess for externalising behaviour problems in children in LMICs, the conceptual frameworks behind the use of these tools in other cultural contexts are not always carefully examined. In order to have valid data across cultures, we should aim to adapt and validate tools before use. Provision of processes to validate tools across LMIC settings would be beneficial.

Administration of autologous bone marrow stem cells into spinal cord injury patients via multiple routes is safe and improves their quality of life: comprehensive case studies.

Presently, there is no cure or effective treatment for spinal cord injury (SCI). Studies in SCI patients have shown that for a treatment to be effective it must primarily improve their quality of life. Numerous studies have shown that stem cells represent an alternative treatment for various disorders and have shown promise in several disease/trauma states. For instance, the use of autologous CD34+ stem cells has been shown to ameliorate symptoms of several disorders such as leukemia, cardiomyopathy, diabetes, and several autoimmune diseases, including multiple sclerosis. For the first time, we report eight case studies of SCI (four acute, four chronic) with approximately 2 years of follow-up that were administered bone marrow stem cells (BMSCs) via multiple routes: directly into the spinal cord, directly into the spinal canal, and intravenous. Magnetic resonance imaging illustrated morphological changes in the spinal cord of some of the patients following BMSCs administration. Comprehensive evaluations demonstrate improvements in ASIA, Barthel (quality of life), Frankel, and Ashworth scoring. Moreover, in order to assess bladder function, we designed a simple numerical clinical scoring system that demonstrates significant changes in bladder function following BMSCs administration. To date, we have administration BMSCs into 52 patients with SCI and have had no tumor formations, no cases of infection or increased pain, and few instances of minor adverse events. These studies demonstrate that BMSCs administration via multiple routes is feasible, safe, and may improve the quality of life for patients living with SCI.

Altered epitope expression of human interstitial fluid apolipoprotein A-I reduces its ability to activate lecithin cholesterol acyl transferase.

In human peripheral interstitial fluid, esterification of cholesterol by lecithin cholesterol acyltransferase (LCAT) was found to occur at a rate of only 10% of that in plasma (5.6 +/- 1.8 compared with 55.6 +/- 7.8 nmol/ml per h). Measurement of cholesterol esterification in the presence of excess reconstituted apoA-I HDL (rA-I HDL) revealed an LCAT activity in interstitial fluid of 24% of that in plasma, indicating that the low rate of esterification could not be caused by limiting mass of LCAT enzyme. When plasma was diluted to the same concentration as in interstitial fluid, the percent cholesterol esterification rate was the same as undiluted plasma and significantly higher than that of interstitial fluid. These findings led us to postulate that poor activation of LCAT in interstitial fluid may result from a change in conformation in apoA-I. To test this hypothesis, a monoclonal antibody AI-11 that inhibits apoA-I activation of LCAT was used to measure apoA-I in interstitial fluid and plasma. Antibody AI-11 recognized interstitial fluid apoA-I poorly, whereas a polyclonal antibody recognized interstitial fluid apoA-I normally. Incubation of antibody AI-11 with high density lipoprotein or rA-I HDL inhibited apoA-I activation of LCAT. We conclude that the altered conformation of apoA-I in interstitial fluid may render it a poor activator of LCAT.

Infectious Complications and Multidrug-Resistant Bacteria in Patients With Hematopoietic Stem Cell Transplantation in the First 12 Months After Transplant.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) has been used as treatment in different hematologic and solid malignancies. The aim of this study was to describe the frequency of infectious complications, microbiology, and outcome in patients undergoing HSCT in Mexico during the pre-engraftment period and the impact on mortality rates at 12 months. METHODS: We conducted a retrospective study of all hematologic malignancies that received HSCT from January 2009 and December 2014, at an oncology reference center. RESULTS: We included 210 patients: 144 autologous (69%) and 66 allogeneic HSCT (31%). There were 184 infections documented in 109 patients; incidence rate was 47.2 per 1000 neutropenia/days and 22.4 per 1000 hospitalization/days. The main infections reported were pneumonia (n = 40, 19%), bloodstream infections (n = 36, 17.1%), and central line-associated bloodstream infections (n = 28, 13.3%). There were 110 bacteria isolated, 31 were multidrug-resistant (26 were extended-spectrum beta-lactamase; Escherichia coli). There were 25 disseminated or complicated viral infections and 20 invasive fungal diseases. Fourteen patients died in the first 30 days (all related to the infectious process). In multivariate analysis leukemia, more than 2 chemotherapy regimens before transplant and pneumonia were related to 12-month mortality rates. CONCLUSIONS: Even though infectious processes are frequent in patients with HSCT, multidrug-resistant bacteria were not as frequent as supposed; however, when these microorganisms are involved, mortality rate is increased. It is important to be alert that patients with pneumonia have a significantly increased mortality risk in the first year.

Antigenic, electrophoretic and binding alterations of human C-reactive protein modified selectively in the absence of calcium.

C-Reactive protein (CRP) is a trace component of normal human serum which has a mol. wt of 105,000 and is composed of five apparently identical subunits arranged in cyclic symmetry. The serum concentration of this protein is known to increase dramatically in response to acute inflammatory or necrotic processes. We report here that in the presence of high concentrations of urea significant antigenic, electrophoretic and binding site modifications of CRP occur selectively in the absence of calcium. CRP treated in this way (designated F-CRP) had a pI of 5.4 and alpha-electrophoretic mobility in contrast to the native molecule which had a pI of 6.4 and gamma-mobility. F-CRP retained a partial antigenic identity to native CRP, displayed decreased intrinsic tryptophan fluorescence, and expressed a new antigenic reactivity. A similar neoantigen was expressed by heating CRP selectively in the absence if calcium (63 degrees C, 5 min). Treatment with guanidinium-HCl or deliberate carbamylation did not produce F-CRP or the expression of the F-antigen. The formation of F-CRP in urea or by heat was prevented by the presence of 0.7 mM or more calcium. CRP treated in this way retained identical characteristics to native CRP. F-CRP chromatographed through Sephadex G-150 in the presence or absence of 6M urea as a protein of apparent mol. wt 75, 000 with no evidence for free CRP subunits. The formation of F-CRP from native CRP resulted in a loss of capacity for calcium-dependent binding to the C-polysaccharide despite the persistence of calcium-independent binding reactivity for polycations. These data suggest that in the presence of sufficient calcium CRP can resist urea- or heat-induced structural denaturation, maintaining antigenic, electrophoretic and binding identity to the native molecule. In the absence of calcium, exposure to urea led to increased electrophoretic mobility and exposure of a new antigenic reactivity, and to alterations in the phosphocholine- but not the polycation-binding sites of the native CRP molecule; this new antigenic reactivity may be of value in further studies on the CRP molecule.

Iberian pig early pregnancy: vascular endothelial growth factor receptor system expression in the maternofetal interface in healthy and arresting conceptuses.

In Iberian pigs, a high conceptus loss occurs during the first 30 days of gestation. Although the exact causes for these losses have not been determined to date, the importance of blood vessel development during early pregnancy has been noted. The aim of this study was to analyze the messenger RNA (mRNA) and protein expression of VEGF-rs (vascular endothelial growth factor, the VEGFR1, and the VEGFR2 receptor system) and elucidate a possible relationship with the conceptus status (healthy or arrested) on gestational Days (gd) 22 and 32. Both mRNA and protein expression for VEGF-rs molecules were consistently expressed in conceptuses and endometrium during the pregnancy period analyzed. In endometrium, a significant increase in VEGF mRNA and VEGFR2 mRNA expression in healthy sites was observed as pregnancy advances (P < 0.001 and P < 0.05, respectively), whereas VEGFR1 mRNA expression was maintained at a constant level. Interestingly, a significantly elevated VEGFR2 mRNA expression (P < 0.05) was observed on gd 22 in endometrium from arrested conceptuses. Furthermore, VEGF mRNA and VEGFR1 mRNA expression in trophoblasts from healthy conceptuses decreased as pregnancy proceeded (P < 0.001). Arrested trophoblasts on gd 32 showed higher VEGFR2 mRNA expression than healthy conceptuses (P < 0.05). Although, in endometrium attachment sites, the pattern of VEGF-rs immunostaning was not affected by conceptus status, the immunoexpression of VEGF-rs in healthy attachment sites increased slightly but consistently as gestation proceeded. In arresting trophoblasts, VEGF and VEGFR2 staining decreased from gd 22 to 32. Moreover, the number of VEGF and VEGFR1-positive capillaries in the subepithelial vascular plexus of endometrium was related to the conceptus status, showing a moderate increase in healthy sites as pregnancy advances. In conclusion, it appears that VEGF-rs is expressed and related to vascular development in Iberian pigs between gd 22 and 32. The upregulated expression of VEGF mRNA and VEGFR2 mRNA in healthy uterine sites suggests a significant role for these angiogenic factors in early pregnancy.

Intervenciones psicológicas en cuidadores primarios de pacientes con trasplante de células hematopoyéticas: Revisión descriptiva / Psychological interventions on primary care healthcare providers of patients with hematopoietic cells transplantation: Descriptive revision / Intervenções psicológicas em cuidadores primários de pacientes com transplante de células hematopoiéticas: Revisão descritiva

Resumen Introducción: El ser cuidador primario informal de un paciente con indicación médica de trasplante de células progenitoras hematopoyéticas puede tener consecuencias negativas en su salud mental y calidad de vida. Objetivo: Describir las intervenciones psicológicas disponibles para el cuidador primario de pacientes sometidos a trasplante de células hematopoyéticas. Metodología: Se realizó una búsqueda sistematizada de los últimos 10 años con los términos MeSH: psychotherapy AND caregive AND stem cell transplantation en las principales bases de datos médicas y de psicología, para su análisis se empleó la estrategia: Problema, Intervención, Comparación y Outcomes (PICO). Resultados: Se identificaron 122 artículos, de ellos diez cumplieron los criterios de inclusión. Las intervenciones provenían de profesionales de enfermería o trabajo social; el 50% incluyó diadas (paciente y cuidador primario), mostraron una tendencia de duración corta, enfocada al periodo posterior al trasplante. Se basan en el entrenamiento en solución de problemas, manejo de estrés, atención plena y expresión emocional. Las intervenciones lograron la disminución de la depresión, ansiedad y estrés en el cuidador; pero no alcanzaron permanencia en la significancia estadística de dichos restablecimientos. Discusión: De acuerdo con lo observado en las publicaciones y por su impacto positivo en la salud mental, se recomienda la implementación de intervenciones psicológicas en cuidadores de pacientes con trasplante de células progenitoras hematopoyéticas. Conclusión: El apoyo psicológico brindado al cuidador generalmente es de profesionales de la salud que no pertenecen al área de la psicología, con resultados clínicos favorables en las etapas más críticas de su estado mental.

Abstract Introduction: Being an informal primary healthcare provider of a patient who undergoes hematopoietic progeny cells transplantation can have adverse consequences on mental health and the quality of life. Objective: To describe the available psychological interventions for the primary healthcare provider of patients undergoing hematopoietic cells transplantations. Methodology: A systematized search of the last 10 years using the MeSH terms psychotherapy AND caregiver AND stem cell transplantation was conducted on the main medical and psychological databases. The analysis strategy followed the PICO scheme (Problem, Intervention, Comparison, Outcomes). Results: 122 articles were identified, and 10 of them fulfilled the inclusion criteria. The interventions were related to nursing or social work professionals. 50% described patient-healthcare provider dyads with short interventions focused on the post-transplantation period. Discussion: According to what has been observed in the publications and due to its positive impact on mental health, the implementation of psychological interventions is recommended in caregivers of patients who underwent hematopoietic stem cell transplantation. Conclusion: The psychological support provided to the caregiver comes mainly from health professionals who do not belong to the area of psychology, with favorable clinical results in the most critical periods for their mental state.

Resumo Introdução: Ser cuidador primário informal de um paciente sometido a transplante de células progenitoras hematopoiéticas pode ter consequências negativas na saúde mental e na qualidade de vida. Objetivo: Descrever as intervenções psicológicas disponíveis para o cuidador primário de pacientes sometidos a transplante de células hematopoiéticas. Metodologia: Realizou-se uma busca sistematizada dos últimos 10 anos com os termos MeSH: psychotherapy AND caregive AND stem cell transplantation nas principais bases de dados médicas e de psicologia, para sua análise realizou-se a estratégia: Problema, Intervenção, Comparação e Outcomes (PICO). Resultados: Identificaram-se 122 artigos, dos quais, dez cumpriram os critérios de inclusão. As intervenções provinham de profissionais em enfermagem ou trabalho social; o 50% incluiu díades (paciente e cuidador primário), mostraram uma tendência de duração curta, focalizada no período posterior ao transplante. Baseiam-se no treinamento em solução de problemas, manejo de estresse, atenção plena e expressão emocional. As intervenções conseguiram melhoras clínicas na diminuição da depressão, ansiedade e estresse no cuidador; mas não alcançaram permanência na significância estatística destes restabelecimentos. Discussão: Conforme o observado nas publicações e por seu impacto positivo na saúde mental, recomenda-se a implementação de intervenções psicológicas em cuidadores de pacientes para quem se indicou transplante de células progenitoras hematopoiéticas. Conclusão: O apoio psicológico oferecido ao cuidador vem de principalmente profissionais da saúde que não pertencem à área da psicologia, com resultados clínicos favoráveis nos períodos mais críticos para seu estado mental.

Epidermal growth factor stimulates integrin-mediated cell migration of cultured human corneal epithelial cells on fibronectin and arginine-glycine-aspartic acid peptide.

PURPOSE: The aim of this work was to show epidermal growth factor (EGF)-dependent migration of human corneal epithelial cells to fibronectin and GRGDSP peptide. The authors assessed the role of cell surface integrin heterodimer alpha 5 beta 1 in mediating haptotactic cell migration to fibronectin by the use of specific function-blocking integrin antibodies. METHODS: A haptotactic cell migration assay in a Boyden chamber was used to compare the relative migration of the cultured human corneal epithelial cells in the presence of fibronectin and GRGDSP peptide-coated filters. Epithelial cells were incubated in the presence of function-blocking integrin antibodies or anti-EGF-receptor antibodies to determine their role in haptotactic cell migration. RESULTS: Human corneal epithelial cells grown as primary cultures migrated in the presence of fibronectin or GRGDSP peptide, but only on stimulation with EGF. Antibodies to the EGF receptor blocked the EGF-mediated stimulation of haptotactic cell migration. Anti-beta 1 and anti-alpha 5 antibodies each inhibited haptotactic cell migration to fibronectin and GRGDSP peptide. CONCLUSIONS: Epidermal growth factor provides an important stimulus of haptotactic cell migration of human corneal epithelial cells. Stimulation of cell migration by EGF was maximal in the range of 5 to 10 ng/ml; this response was completely blocked by incubation with an anti-EGF receptor antibody. Function-blocking integrin antibodies, specifically anti-beta 1 and anti-alpha 5, inhibited integrin-mediated cell migration to fibronectin and GRGDSP peptide. These data suggest that EGF represents an essential initial stimulus for haptotactic cell migration of human corneal epithelial cells; furthermore, integrins are important in mediating cell migration to fibronectin and GRGDSP:

Involvement of integrins with adhesion-promoting, heparin-binding peptides of type IV collagen in cultured human corneal epithelial cells.

PURPOSE: The aim of this work was to identify the integrin subunits present on the cell surface of human corneal epithelial cells. The authors determined to show whether type IV collagen, heparin-binding peptides of type IV collagen (Hep-I, Hep-II, and Hep-III), fibronectin, and GRGDSP promote cell adhesion of human corneal epithelial cells. Type IV collagen and heparin-binding peptides of type IV collagen may be important in corneal epithelial cell adhesion in normal and pathologic conditions and reepithelialization. The authors assess the role of cell surface integrins in mediating cell adhesion to these proteins and peptides. METHODS: Fluorescence-activated cell sorter (FACS) analysis was used to determine the integrin subunits expressed at the cell surface of the cultured human corneal epithelial cells. Cell adhesion was assessed with type IV collagen, heparin-binding peptides of type IV collagen, fibronectin, and GRGDSP: Antibodies to the integrin subunits were used to determine the potential role of integrins in cell adhesion to the above proteins and peptides. RESULTS: FACS analysis identified the beta 1, beta 4, alpha 2, alpha 3, alpha 5, alpha 6, and alpha v integrin subunits on human corneal epithelial cells grown as primary cultures. The anti-beta 1 antibody inhibited cell adhesion to heparin-binding peptides of type IV collagen, type IV collagen, fibronectin, and GRGDSP: Antibodies to the alpha 2 integrin subunit inhibited cell adhesion to the heparin-binding peptides of type IV collagen and slightly inhibited cell adhesion to intact type IV. Antibodies to the alpha 3 integrin subunit exhibited a somewhat lesser effect compared to the anti-alpha 2 integrin antibody. CONCLUSIONS: These data show that the alpha 2 beta 1 integrin of human corneal epithelial cells recognize heparin-binding peptide sequences derived from human type IV collagen. It seems likely that these sequences play an important role in integrin-mediated corneal epithelial cell adhesion. In addition, the alpha 3 beta 1 integrin may mediate similar events.

Adenovirus-mediated in vivo gene transfer in a rabbit model of allograft vasculopathy.

BACKGROUND: The long-term success of heart transplantation continues to be in jeopardy because of the development of accelerated vascular myointimal proliferation. Transfer of genes encoding products that can modulate the adverse consequences of phenomena that cause myointimal proliferation, into the allograft vessel wall, may modify these pathologic processes. The purpose of this study was to assess the feasibility of gene transfer and to evaluate the duration of gene expression in a rabbit heterotopic aortic transplant model of allograft vasculopathy. METHODS: The abdominal aortas of 32 outbred New Zealand rabbits were harvested and cross-sectionally bisected (n = 64). Six donor and recipient animals were used in a preliminary study to examine neointimal proliferation without accompanying gene transfer. Of the remaining 26 rabbits (52 allografts), one half of each allograft aorta was administered a control solution, while the other half was incubated with a replication-defective, recombinant, adenoviral vector-encoding, cytomegalovirus promoter-regulated beta-galactosidase. After a 20-minute incubation period, bilateral aorto-carotid transplantations were performed in 26 recipient rabbits. All animals received cyclosporine immunosuppression (10 mg/kg/day subcutaneously). The allografts were harvested at 3, 7, 10, 21, and 28 days after transplantation and assayed for beta-galactosidase activity. RESULTS: Neointimal areas showed an initially slow increase for the first 10 days, followed by a rapid increase up to 21 days, and tended to plateau thereafter. Significant beta-galactosidase was apparent in aortic sections dissected from host rabbits for all time points, except at 28 days. At the 21-day time point, the aortic section from one rabbit was positive, whereas the other two remained negative. However, the one positive section showed intense beta-galactosidase activity, suggesting variability in the experimental model. At 28 days, all aortic sections were negative. CONCLUSIONS: Our findings confirm that genes delivered by this method are expressed for the duration of early rapid intimal proliferation in this heterotopic rabbit model of aortic allograft vasculopathy. These findings suggest that this animal model can be used to assess the therapeutic potential of gene transfer at the time of vascular transplantation and may provide a novel therapeutic approach to prevent or ameliorate the genesis of allograft vasculopathy.

Absence of normal keratan sulfate in the blood of patients with macular corneal dystrophy.

We measured levels of sulfated keratan sulfate in serum using a monoclonal antibody in an enzyme-linked immunosorbent assay. Sulfated keratan sulfate was not detected in the serum of 16 patients with macular corneal dystrophy, but was present at normal levels in 66 patients with other corneal diseases. There were no differences with respect to age, sex, and other ocular findings. This monoclonal antibody recognizes a sulfated carbohydrate epitope present in both corneal and skeletal keratan sulfate. Since most serum keratan sulfate is derived from the cartilages, the defect in keratan sulfate synthesis in macular corneal dystrophy may not be restricted to corneal cells. This assay should prove useful in the diagnosis of macular corneal dystrophy, particularly in children at risk before the appearance of opacification.

Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres.

Adult, canine intervertebral disc cells were isolated with a sequential digestion of pronase and bacterial collagenase. The nonchondrodystrophoid nucleus pulposus exhibits two populations of cells: large notochordal cells and smaller chondrocyte-like cells. The cells from the transition zone and anulus fibrosus are uniform in size, ranging from 17 to 21 microns. The isolated cells were encapsulated in alginate beads and cultured in Ham's F-12 medium containing 5% heat-inactivated fetal bovine serum. Alginate bead formation requires calcium ions and can be reversed with a suitable chelator, thus releasing viable cells. We observed that 58% of the newly synthesized proteoglycans formed large-molecular-weight aggregates with hyaluronic acid. The proteoglycans contained low amounts of keratan sulfate (KS) (less than 5% of the total glycosaminoglycans synthesized). The chondroitin sulfates (CS) consisted of 51-67% as 6-O-sulfate and 29-39% as 4-O-sulfate, with the remainder (4-10%) present as 4,6-sulfate for all three zones of the disc. The majority of cells synthesized significant amounts of matrix as evidenced by Alcian Blue staining. By immunohistochemical analysis, the matrix contained chondroitin 6-sulfate as demonstrated by monoclonal antibodies to the unsaturated disaccharides remaining on the proteoglycan core after chondroitinase ABC digestion. Keratan sulfate was also present in the majority of the matrices around cells. These results emphasize the similarity of the newly synthesized proteoglycans secreted by cells grown in alginate beads to those synthesized by the neonate disc. These experiments also demonstrate the usefulness of this method as a microculture technique for disc cells.

Clonal analysis of the population of chondrocytes from the Swarm rat chondrosarcoma in agarose culture.

Chondrocytes from the Swarm chondrosarcoma, a transplantable rat tumor, have been difficult to maintain in tissue culture for extended periods due to a time-dependent alteration of the culture to a more fibroblastic phenotype. This feature precluded the use of these cultures to examine chronic conditions that may affect cell metabolism, and the homogeneity or heterogeneity of the tumor cells within the culture population could not be examined. Use of suspension culture in agarose stabilized the chondrocyte phenotype, permitting long-term culture. Clones of tumor chondrocytes were established in agarose and were examined over 2-3 weeks for evidence that the cells were accumulating a proteoglycan-rich extracellular matrix, as determined by positive staining by Alcian blue, and were undergoing cell division. Nearly 90% of the cloned cells exhibited a prominent extracellular matrix by day 7 of culture and greater than 99% did so by day 14. Cell division did not occur to any great extent until days 6-7 of culture. After this lag, the cells appeared to undergo logarithmic growth, with a cell generation time of about 12 days. By 20 days of culture, between 80 and 90% of the initial clones contained multiple cells, indicating that nearly all the cells were in, or had entered, the cell cycle. These results suggest that the chondrocytes from the rat chondrosarcoma form a homogeneous cell population with respect to their ability to synthesize an extra-cellular matrix and divide.

Procedure for development of an enzyme-linked immunosorbent assay. Development of an assay for human apolipoprotein A-I.

A set of criteria for selection of antibodies during the development of enzyme-linked immunosorbent assay (ELISA) is described. Using these criteria, a competitive ELISA for human apo A-I using a polyclonal goat antibody was developed. The assay recognizes apo A-I from plasma and high-density lipoprotein (HDL), as well as the pure delipidated apo A-I, equally. Intra- and inter-assay variations were 5.2% and 3.5%, respectively. Recovery rate, as determined by spiking a known quantity of pure delipidated apo A-I into a reference plasma, was determined to be 101.3%. The assay was validated by comparing the concentration of apo A-I in HDL with the dye elution method. The apo A-I ELISA to apo A-I dye elution ratio was 1.01. Apo A-I concentration in Centers for Disease Control reference material determined by this method was in agreement with the reported consensus value. Repeated freezing and thawing of the samples (three freeze-thaw cycles at -20 degrees C) as well as long-term freezing (up to 1 year at -70 degrees C) did not affect the concentration of apo A-I in the samples. The assay was applicable both to normolipidemic and dyslipidemic plasmas. No immunologic difference was noted when plasma from dyslipidemic subjects was assayed. A frequent problem of long-term storage is deamidation. The values found for apo A-I in a deamidated plasma were the same as those for the corresponding fresh plasma. Plasma apo A-I values were also positively correlated with that of HDL-cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)