A microaliquoting technique for precise histological annotation and optimization of cell content in frozen tissue specimens.

Knowledge of the exact cell content of frozen tissue samples is of growing importance in genomic research. We developed a microaliquoting technique to measure and optimize the cell composition of frozen tumor specimens for molecular studies. Frozen samples of 31 mesothelioma cases were cut in alternating thin and thick sections. Thin sections were stained and evaluated visually. Thick sections, i.e., microaliquots, were annotated using bordering stained sections. A range of cellular heterogeneity was observed among and within samples. Precise annotation of samples was obtained by integration and compared to conventional single face and "front and back"' section estimates of cell content. Front and back estimates were more highly correlated with block annotation by microaliquoting than were single face estimates. Both methods yielded discrepant estimates, however, and for some studies may not adequately account for the heterogeneity of mesothelioma or other malignancies with variable cellular composition. High yield and quality RNA was extracted from precision annotated, tumor-enriched subsamples prepared by combining individual microaliquots with the highest tumor cellularity estimates. Microaliquoting provides accurate cell content annotation and permits genomic analysis of enriched subpopulations of cells without fixation or amplification.

Congenital disorder of glycosylation type Ia in a 6-year-old girl with a mild intellectual phenotype: two novel PMM2 mutations.

We report two novel mutations in the PMM2 gene in a girl with congenital disorder of gylcosylation type Ia (CDG Ia) and a mild intellectual phenotype.

Participación de organizaciones comunitarias en la gestión de salud: una evaluación de la experiencia del Programa UNI / Community participation in health management: an evaluation of the UNI Program experience

Com o propósito de melhorar a formação dos profissionais da saúde na América Latina, a Fundação W.K.Kellogg convocou as universidades de vários países para apresentar projetos que integram as respectivas faculdades da área da saúde, os serviços públicos de saúde da área de influencia e as organizações comunitárias que aí atuarão. Assim, ao longo da década de 1990 desenvolveu-se o Programa UNI (Uma Nova Iniciativa na Educação dos Profissionais da Saúde: União com a Comunidade). Este artigo concentra seu olhar no componente comunitário desses projetos, baseado nos resultados obtidos no Estudo Especial de Comunidade realizado no marco do Programa de Apoio aos projetos UNI. Um aspecto central e decisivo do programa UNI tem sido o esforço de cooperação entre três atores: Universidades, Serviços de Saúde e Comunidade, que se associaram em parcerias para apoiar processos de mundanças paralelas nas instituições de cada ator social e nas cidades em que cada projeto foi executado. A avaliação realizada colocou o eixo da observação na "borda da população" e suas organizações, tentando identificar as condições que facilitem a construção da cidadania, a imagem de superação intergeneracional que podem alcançar os membros da comunidade, e as mudanças nas condições de vida, as três dimensões consideradas pela comunidade como relevantes para serem avaliadas.

The alpha folate receptor is highly activated in malignant pleural mesothelioma.

OBJECTIVE: To determine whether the folate receptor gene is overexpressed in malignant pleural mesothelioma. METHODS: Differential display analysis was performed with fresh frozen RNA obtained from normal lung, pleura, and mesothelioma. Sixty differentially expressed genes were identified and characterized. One gene that was over-expressed in mesothelioma versus normal tissue was the human alpha folate receptor. In situ hybridization with antisense probes designed on the basis of the sequence of the folate receptor was performed with frozen sections from 61 patients (33 epithelial and 28 mixed or sarcomatoid tumors) with malignant pleural mesothelioma. The controls included normal pleura, normal lung, other cancers, and sense controls for all of the tumors. Northern analysis with a folate receptor probe and immunohistochemical analysis with anti-alpha folate receptor antibodies were also performed. RESULTS: Forty-four (72%) of the 61 mesothelioma tumors were found to have between 2-fold and 4-fold higher mRNA expression of the folate receptor when compared with the control tissues. The histologic type of the tumor did not affect the rate of folate receptor activation. Northern analysis and immunohistochemical experiments confirmed these findings. CONCLUSIONS: A majority of mesothelioma tumors examined overexpress the alpha folate receptor protein when compared with normal adjacent tissues. This finding may help explain the observations that antifolate drugs have activity in the treatment of mesothelioma. It also encourages further study of folate receptor-related treatment strategies in this malignancy.

Cell motility as a prognostic factor in Stage I nonsmall cell lung carcinoma: the role of gelsolin expression.

BACKGROUND: Tumor cell motility is an important characteristic that facilitates the multistep process of tumor metastasis. Rac, ABP-280, and gelsolin are proteins that interact with actin and are important in cell motility. METHODS: The authors studied a cohort of 229 Stage I nonsmall cell lung carcinoma (NSCLC) patients who had a minimum of 3 years follow-up and had been previously analyzed for 22 clinical, pathologic, and molecular features, of which 9 had been found to provide significant prognostic information in a Cox proportional hazards model. Tumor sections were stained by the avidin-biotin complex method using monoclonal antibodies against rac, ABP-280, and gelsolin. RESULTS: In a pilot analysis of over 50 patients each, rac and ABP-280 were found to be moderately-to-highly expressed in the majority of tumors and to provide no prognostic information. Gelsolin expression was more variable and appeared to be negatively correlated with survival in the pilot population. In the larger 229-patient population, high focal gelsolin expression was seen in 32 tumors (14%) and conferred the highest relative risk (4.04) of cancer recurrence among all factors tested, compared with tumors that had no or low gelsolin expression. Moderate focal gelsolin expression, seen in 46 patients (20%), also conferred a significant risk of cancer recurrence, with a relative risk of 2.26 compared with tumors that had no or low gelsolin expression. Consideration of average gelsolin expression and of overall survival yielded similar results. CONCLUSIONS: Gelsolin expression appears to be a significant prognostic factor for cancer recurrence in cases of Stage I NSCLC.

A follow-up study of infants < or = 2000 g birthweight treated in central Queensland.

OBJECTIVE: This follow-up study was undertaken in an effort to ascertain the morbidity in the survivors of infants < or = 2000 g birthweight cared for in the two Rockhampton intensive care nurseries. METHODOLOGY: The records of all infants < or = 2000 g delivered in or transferred to Rockhampton during the 11 year period 1979 through 1989 inclusive were extracted. Efforts were made to contact and examine all of these children. Those found to be disabled were assessed as being mildly, moderately or severely affected. RESULTS: Of the 482 infants of birthweight < or = 2000 g treated in the period under review, 393 survived to be discharged from hospital. Eight were known to have died subsequently. Of the remaining 385 children, 288 (74.8%) were able to be contacted and their health status determined. A total of 36 infants were found to have significant disabilities. Twenty-four were mildly affected, five moderately and seven severely affected. Severe disability in infants of < or = 1000 g was 16% (3/19). CONCLUSIONS: The incidence of disability was established in 74.8% of the surviving population. It was not dissimilar to the incidence of disability in similar birthweight groups in some Australian tertiary centres for the years under study. It is emphasized that the follow-up was incomplete and recognized that the survival rates and incidence of disability in survivors has improved in tertiary centres since the time frame of this study.

Stimulation of goblet cell mucous secretion by activation of nerves in rat conjunctiva.

An epithelial debridement wound, as a stimulus to the cornea, causes conjunctival goblet cell mucous secretion in that eye. To determine if this stimulation of secretion is neurally mediated, rats were anesthetized and the local anesthetic lidocaine (1%) or buffer alone was administered topically and/or subconjunctivally for 15 min. A corneal epithelial debridement wound was made in one eye. The contralateral eye served as the control. After 5-120 min, animals were sacrificed and inferior bulbar conjunctival buttons removed. Mucus in the goblet cells was stained with Alcian blue and periodic acid-Schiff's reagent to indicate mucin-containing goblet cells. The number of mucin-containing goblet cells/0.16 mm2 was determined by light microscopy; a decrease in number indicated an increase in mucous secretion. Stimulation by corneal wounding induced goblet cell mucous secretion in that eye. Secretion was observed as rapidly as 5 min after stimulus and for as long as 120 min. Topical application of lidocaine, subconjunctival injection of lidocaine, or a combination of both inhibited wound-induced stimulation of mucous secretion. We conclude that conjunctival goblet cell mucous secretion can be neurally mediated and could serve as an immediate response to protect the ocular surface.

Localization of nerves adjacent to goblet cells in rat conjunctiva.

Neural stimulation of the cornea induces conjunctival goblet cell mucous secretion. Immunofluorescence microscopy was used to determine if nerves are present near conjunctival goblet cells and what types of nerves are present. In euthanized rats, the local anesthetic lidocaine (1%) was placed topically on the ocular surface for 10 min to prevent goblet cell mucous secretion. The ocular surface tissues were removed and either fixed in formaldehyde and then frozen, or frozen first and then post-fixed in formaldehyde. Tissue was sectioned and nerves localized by indirect immunofluorescence microscopy, using antibodies to synaptophysin (indicates nerve, independent of type), vasoactive intestinal peptide (VIP, indicates parasympathetic nerves), tyrosine hydroxylase (TH, indicates sympathetic nerves), dopamine beta-hydroxylase (DBH, indicates sympathetic nerves), phenylethanolamine-N-methyltransferase (PNMT, indicates sympathetic nerves), and calcitonin gene-related peptide (CGRP, indicates sensory nerves). Goblet cells were identified by phase-contrast microscopy. Synpatophysin-containing nerves were present in the basolateral region of conjunctival goblet cells clusters. Nerve fibers immunoreactive to VIP were found in the conjunctiva along the epithelial-stromal junction and around the basolateral aspect of goblet cell clusters. Nerve fibers immunoreactive to TH and DBH were detected surrounding goblet cells and in the conjunctival stroma. Nerve fibers immunoreactive to CGRP were detected in the epithelium and at the epithelial stromal junction, but were not localized near goblet cell clusters. CGRP-containing nerve fibers were also detected in the conjunctival stroma under the epithelium. We conclude that efferent parasympathetic and sympathetic, but not afferent sensory, nerves appear to be located adjacent to conjunctival goblet cell clusters. Activation of efferent parasympathetic and sympathetic nerves could directly stimulate conjunctival goblet cell mucous secretion. Antidromic activation of afferent sensory nerves releasing neurotransmitters could stimulate goblet cell secretion by a paracrine mechanism.

Survival of low birthweight infants in central Queensland.

The survival of low birthweight infants < or = 2000 g born in the central Queensland area during the years 1979 to 1991 was examined. Five hundred and sixty such infants were either delivered at one of the two Rockhampton obstetric units (Rockhampton Base Hospital and the Mater Misericordiae Hospital) or retrieved from outlying central Queensland areas. Both hospitals had intensive care nurseries capable of ventilation and infants were transferred to tertiary centres only for cardiac or surgical treatment. The study involved all liveborn infants, including those with lethal malformations and all deaths up to the time of discharge. Survival rates were as follows: < or = 500 g 0%, 501 < or = 750 g 30%, 751 < or = 1000 g 51%, 1001 < or = 1500 g 79%, 1501 < or = 2000 g 93%. The survival of Aboriginal infants and outborn infants were found to be essentially the same as the overall group.

Undergraduate orthodontics--a collision course on occlusion.

Five years ago the dental school at Belfast instituted a 'longitudinal course' in orthodontics. It has meant changes to attitudes and working practices, and staff and students have been both flexible and open minded. Here, Andrew Richardson and Hill Mercer, outline the course, the contortions, and some of the problems that have been straightened out.