Fragilidade em idosos que vivem na comunidade: prevalência e fatores associados / Frailty in community-dwelling older people: prevalence and associated factors

Fundamentos: Idosos que vivem na comunidade são propensos a desenvolver fragilidade, considerada como um estado clinicamente identificável que aumenta a vulnerabilidade a resultados adversos e prediz incapacidade e mortalidade na população idosa. Objetivo: Identificar a prevalência e fatores associados à fragilidade em idosos que vivem em uma comunidade. Método: Trata-se de um inquérito domiciliar transversal e analítico, de abordagem quantitativa realizado com 854 idosos que vivem na comunidade. A fragilidade foi mensurada pela Edmonton Frail Scale (EFS). A associação entre fragilidade e variáveis sociodemográficas e de condições clínicas foi mensurada pela análise múltipla por regressão logística. Resultados: A prevalência de fragilidade encontrada neste estudo foi de 12,3% (IC95%: 10,1 a 14,5). O modelo de regressão logística mostrou que as variáveis estatisticamente associadas à fragilidade foram: queda recorrente, uso de dispositivo para auxílio à marcha, polifarmácia, autopercepção ruim de saúde, dependência nas atividades básicas e instrumentais de vida diária. Conclusão: A prevalência de fragilidade em idosos foi baixa em comparação a outros estudos nacionais que empregaram a EFS. Os resultados indicaram múltiplos fatores associados à fragilidade modificáveis. Assim, a investigação da síndrome da fragilidade bem como dos seus fatores relacionados passíveis de prevenção são ações a serem incluídas na prática clínica. (AU)

Foundations: Elderly people living in the community are prone to developing frailty, considered as a clinically identifiable state that increases vulnerability to adverse events and predicts disability and mortality in the elderly population. Objective: To identify the prevalence and factors associated with frailty in the elderly living in the community. Materials and method: This is a cross-sectional and analytical household survey with a quantitative approach conducted with 854 elderly people living in the community. Frailty was measured by Edmonton Frail Scale (EFS). The association between frailty and sociodemographic and clinical condition variables was measured by multiple analysis using logistic regression. Results: The prevalence of frailty found in this study was 12.3% (95% CI: 10.1 to 14.5). The logistic regression model showed that the variables statistically associated with frailty were: recurrent fall, use of walking aids, polypharmacy, poor self-rated health, dependence on basic and instrumental activities of daily living. Conclusion: The prevalence of frailty in the elderly was low compared to other national studies that employed the SAI. Results indicated potentially modifiable factors associated with frailty. Thus, the investigation of frailty syndrome and its related preventable factors are actions to be included in clinical practice. (AU)

Study protocol for a randomized controlled trial on the effect of the Diabetic Foot Guidance System (SOPeD) for the prevention and treatment of foot musculoskeletal dysfunctions in people with diabetic neuropathy: the FOotCAre (FOCA) trial I.

BACKGROUND: This study is part of a series of two clinical trials. Taking into account the various musculoskeletal alterations of the foot and ankle in people with diabetic peripheral neuropathy (DPN) and the need for self-care to avoid more serious dysfunctions and complications, a self-manageable exercise protocol that focuses on strengthening the foot muscles is presented as a potentially effective preventive method for foot and gait complications. The aim of this trial is to investigate the effect of a customized rehabilitation technology, the Diabetic Foot Guidance System (SOPeD), on DPN status, functional outcomes and gait biomechanics in people with DPN. METHODS/DESIGN: Footcare (FOCA) trial I is a randomized, controlled and parallel two-arm trial with blind assessment. A total of 62 patients with DPN will be allocated into either a control group (recommended foot care by international consensus with no foot exercises) or an intervention group (who will perform exercises through SOPeD at home three times a week for 12 weeks). The exercise program will be customized throughout its course by a perceived effort scale reported by the participant after completion of each exercise. The participants will be assessed at three different times (baseline, completion at 12 weeks, and follow-up at 24 weeks) for all outcomes. The primary outcomes will be DPN symptoms and severity classification. The secondary outcomes will be foot-ankle kinematics and kinetic and plantar pressure distribution during gait, tactile and vibration sensitivities, foot health and functionality, foot strength, and functional balance. DISCUSSION: As there is no evidence about the efficacy of rehabilitation technology in reducing DPN symptoms and severity or improving biomechanical, clinical, and functional outcomes for people with DPN, this research can contribute substantially to clarifying the therapeutic merits of software interventions. We hope that the use of our application for people with DPN complications will reduce or attenuate the deficits caused by DPN. This rehabilitation technology is freely available, and we intend to introduce it into the public health system in Brazil after demonstrating its effectiveness. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04011267. Registered on 8 July 2019.

Pulmonary actinomycosis masquerading as lung cancer: A case report.

Pulmonary actinomycosis is a rare yet important and challenging diagnosis to make. It is commonly confused with other lung diseases, such as tuberculosis and bronchogenic carcinoma, leading to delay diagnosis or misdiagnosis. A 49-year-old man presented with a chronic cough, hemoptysis, and pleuritic chest pain. His initial imaging studies including computed tomography (CT) was suggestive of bronchogenic carcinoma. A subsequent CTguided biopsy was consistent with pulmonary actinomycosis and excluded the possibility of bronchogenic carcinoma. He was treated with antibiotic therapy and achieved remission with complete radiological resolution upon follow-up.

The effect of different photoperiods in circadian rhythms of per3 knockout mice.

The aim of this study was to analyse the circadian behavioural responses of mice carrying a functional knockout of the Per3 gene (Per3(-/-)) to different light : dark (L : D) cycles. Male adult wild-type (WT) and Per3(-/-) mice were kept under 12-hour light : 12-hour dark conditions (12L : 12D) and then transferred to either a short or long photoperiod and subsequently released into total darkness. All mice were exposed to both conditions, and behavioural activity data were acquired through running wheel activity and analysed for circadian characteristics during these conditions. We observed that, during the transition from 12L : 12D to 16L : 8D, Per3(-/-) mice take approximately one additional day to synchronise to the new L : D cycle compared to WT mice. Under these long photoperiod conditions, Per3(-/-) mice were more active in the light phase. Our results suggest that Per3(-/-) mice are less sensitive to light. The data presented here provides further evidence that Per3 is involved in the suppression of behavioural activity in direct response to light.

Physical therapy intervention (PTI) increases plasma brain-derived neurotrophic factor (BDNF) levels in non-frail and pre-frail elderly women.

Biomarkers are important factors in the identification of the frail elderly (higher risk of developing disease) and in assessing the impact of PTI. On the other hand, BDNF has been related to neuroprotection in a series of central nervous system diseases in older age. The levels of BDNF in groups of elderly women classified according to Fried phenotype (non-frail and pre-frail) were compared. We assessed the impact of a PTI on BDNF levels. A convenience sample of 48 elderly women was randomly selected. The PTI group was composed by 20 elderly women selected from this group. Plasma neurotrophic factors, such as BDNF, glial-derived neutrophic factor (GDNF), and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA). Timed-up-and-go (TUG) test, hand-grip and work/body weight were evaluated before and after the intervention. Plasma concentrations of BDNF were significantly higher in non-frail in comparison to pre-frail elderly women. After the PTI, higher levels of BDNF were found in elderly women (before 351±68 pg/ml and after 593±79 pg/ml; p<0.001). Both groups had an increase in BDNF levels after the PTI. The low levels of BDNF in pre-frail elderly women suggest that this neurotrophic factor may be a key pathophysiological mediator in the syndrome of frailty. The fact that PTI increased BDNF levels in both groups suggests that it may be possible to modify this phenotype.

Effects of 174 G/C polymorphism in the promoter region of the interleukin-6 gene on plasma IL-6 levels and muscle strength in elderly women

We investigated the effect of -174 G/C single-nucleotide polymorphism in the promoter region of the IL6 gene on plasma IL-6 levels and muscle strength, and the relationship between IL-6 levels and muscle strength in elderly women. The sample consisted of 199 elderly residents (73.0 ± 7.8 years old) from rest homes and the community in Belo Horizonte, MG, Brazil. -174 G/C polymorphism was determined by direct sequencing of the product by PCR, and plasma IL-6 concentrations were measured by ELISA. Muscle strength in the knee joint was evaluated using a Biodex System 3 Pro® isokinetic dynamometer. ANCOVA was used to determine the effect of polymorphism on IL-6 levels and muscle strength, and the Pearson correlation coefficient to assess the relationship between IL-6 levels and muscle strength. -174 G/C polymorphism was associated with the plasma IL-6 levels of elderly women (P < 0.01) since homozygotes for the G allele showed high IL-6 levels (GG 3.85 pg/mL, GC + CC 2.13 pg/mL). There was no association of polymorphism on muscle strength (P > 0.05). No association was found between IL-6 levels and knee extensor muscle (r = 0.087, P = 0.306) or flexor (r = -0.011, P = 0.894) strength. An interaction between -174 G/C polymorphism and housing conditions of the sample of elderly women was identified, with the effect of genotype on IL-6 levels being higher in the institutionalized elderly. These results support the evidence that -174 G/C polymorphism of the IL6 gene associates with individual variability of plasma IL-6 levels in elderly women.

Effects of 174 G/C polymorphism in the promoter region of the interleukin-6 gene on plasma IL-6 levels and muscle strength in elderly women.

We investigated the effect of -174 G/C single-nucleotide polymorphism in the promoter region of the IL6 gene on plasma IL-6 levels and muscle strength, and the relationship between IL-6 levels and muscle strength in elderly women. The sample consisted of 199 elderly residents (73.0 ± 7.8 years old) from rest homes and the community in Belo Horizonte, MG, Brazil. -174 G/C polymorphism was determined by direct sequencing of the product by PCR, and plasma IL-6 concentrations were measured by ELISA. Muscle strength in the knee joint was evaluated using a Biodex System 3 Pro® isokinetic dynamometer. ANCOVA was used to determine the effect of polymorphism on IL-6 levels and muscle strength, and the Pearson correlation coefficient to assess the relationship between IL-6 levels and muscle strength. -174 G/C polymorphism was associated with the plasma IL-6 levels of elderly women (P < 0.01) since homozygotes for the G allele showed high IL-6 levels (GG 3.85 pg/mL, GC + CC 2.13 pg/mL). There was no association of polymorphism on muscle strength (P > 0.05). No association was found between IL-6 levels and knee extensor muscle (r = 0.087, P = 0.306) or flexor (r = -0.011, P = 0.894) strength. An interaction between -174 G/C polymorphism and housing conditions of the sample of elderly women was identified, with the effect of genotype on IL-6 levels being higher in the institutionalized elderly. These results support the evidence that -174 G/C polymorphism of the IL6 gene associates with individual variability of plasma IL-6 levels in elderly women.

Influence of socio-demographic, clinical and functional factors on the severity of dementia.

Dementia is one of the most relevant illnesses due to its functional impact on the elderly. The aim of the present study was to determine the influence of socio-demographic, clinical, cognitive and functional factors on the severity of dementia in elderly individuals. One hundred six elderly individuals with a diagnosis of dementia as determined by the DSM-IV/APA participated in the study. Cognition was assessed by the mini-mental state examination (MMSE); functional performance of basic and instrumental activities of daily living (BADLs and IADLs) was assessed by the Katz index (KI), Lawton-Brody index (LBI) and Pfeffer index (PI). The severity of dementia was established through the clinical dementia rating (CDR). In order to determine the influence of factors on the degree of dementia, ordinal regression analysis was performed. Age, MMSE, KI, LBI and PI scores had statistically significant associations to the severity of dementia. However, only the age (odds ratio (OR) = 0.197; confidence interval (CI) = 0.060-0.643) and performance in IADLs (LBI: OR = 1.237, CI = 1.077-1.422; PI: OR = 0.641, CI = 0.548-0.750) were maintained in the final ordinal regression model, R(2) = 0.818. The results show that elderly individuals over 80 years of age and those with a more compromised performance in IADLs have a greater chance of exhibiting more severe degrees of dementia. These findings suggest that the ability to perform IADLs may be an important variable in differentiating degrees of the severity of dementia.

Muscle strength, muscle balance, physical function and plasma interleukin-6 (IL-6) levels in elderly women with knee osteoarthritis (OA).

An increased circulating level of inflammatory cytokines has been associated with sarcopenia, functional disability, chronic diseases, and mortality in the elderly. Osteoarthritis (OA) is a disease common to this population, the seriousness of articular degeneration has been associated to the increase in some cytokines, IL-6 among them. The aim of the present study was to correlate IL-6 plasma levels with muscle strength, endurance, muscle balance hamstring/quadriceps (H/Q) and physical function in 80 elderly women (71.2 ± 5.3) with knee OA. IL-6 was measured using enzyme-linked immunosorbent assay (ELISA). Quadriceps and hamstring muscle strength, endurance and hamstring-quadriceps muscle balance were assessed using a Biodex system 3 pro(®) isokinetic dynamometer. Physical function was assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Spearman's correlation coefficient was used to explore the relationship between the outcomes at the significance level of α=0.05. IL-6 was inversely correlated to the endurance of the hamstring muscles (r=-0.232; p=0.03) and muscle balance (H/Q) on the lower right side at 180°/s (r=-0.254; p=0.023). No significant correlation between IL-6, muscle strength and physical function was found. Our results show that elevated levels of IL-6 may possibly contribute to the reduction of the endurance of hamstring muscles and H/Q muscle balance in the elderly studied.

sTNFR-1 is an early inflammatory marker in community versus institutionalized elderly women.

OBJECTIVE AND DESIGN: To evaluate plasma sTNFR-1 and IL-6 levels and correlate with hand grip in the institutionalized and community living Brazilian elderly. MATERIAL: A convenience sample of 110 elderly women (71.17 + or - 7.44 years) was selected. Plasma sTNFR-1 and IL-6 levels were measured by ELISA. For the measurement of hand grip, a JAMAR dynamometer was used. RESULTS: Plasma concentrations of inflammatory markers were significantly higher in institutionalized elderly (sTNFR-1: 479 + or - 22 pg/mL; IL-6: 6.3 + or - 0.8 pg/mL) than in community-dwelling elderly (sTNFR-1: 329 + or - 24 pg/mL; IL-6: 2.5 + or - 0.4 pg/mL; P < 0.0001). Institutionalized elderly had reduced hand grip (15 + or - 0.8 Kgf) in comparison to community dwelling elderly (23 + or - 0.6 Kgf; P < 0.05). When individuals were subdivided in age groups, sTNFR-1 was higher in community dwelling versus institutionalized elderly in the 60-70 age range. CONCLUSIONS: Our results demonstrate that being institutionalized has an impact on levels of inflammatory markers.

Muscle strength but not functional capacity is associated with plasma interleukin-6 levels of community-dwelling elderly women

The association of plasma interleukin-6 (IL-6) levels, muscle strength and functional capacity was investigated in a cross-sectional study of community-dwelling elderly women from Belo Horizonte, Brazil. Elderly people who present controlled chronic diseases with no negative impact on physical, psychosocial and mental functionality are considered to be community-dwelling. Psychological and social stress due to unsuccessfully aging can represent a risk for immune system disfunctions. IL-6 levels, isokinetic muscle strength of knee flexion/extension, and functional tests to determine time required to rise from a chair and gait velocity were measured in 57 participants (71.21 ± 7.38 years). Serum levels of IL-6 were measured in duplicate and were performed within one single assay (mouse monoclonal antibody against IL-6; High-Sensitivity, Quantikine®, R & D Systems, USA; intra-assay coefficient of variance = 6.9-7.4 percent; interassay coefficient of variance = 9.6-6.5 percent; sensitivity = 0.016-0.110 pg/mL; mean = 0.039 pg/mL). Muscle strength was assessed with the isokinetic dynamometer Biodex System 3 Pro®. After the Shapiro-Wilk normality test was applied, correlations were investigated using Spearman and Kruskal-Wallis tests. Post hoc analysis was performed using the Dunn test. A significant negative correlation was observed between plasma IL-6 levels (1.95 ± 1.77 pg/mL) and muscle strength for knee flexion (70.70 ± 21.14 percent; r = -0.265; P = 0.047) and extension (271.84 ± 67.85 percent; r = -0.315; P = 0.017). No significant correlation was observed between IL-6 levels and the functional tests (time to rise from a chair = 14.65 ± 2.82 s and gait velocity = 0.95 ± 0.14 m/s). These results suggest that IL-6 is associated with reduced muscle strength.

Muscle strength but not functional capacity is associated with plasma interleukin-6 levels of community-dwelling elderly women.

The association of plasma interleukin-6 (IL-6) levels, muscle strength and functional capacity was investigated in a cross-sectional study of community-dwelling elderly women from Belo Horizonte, Brazil. Elderly people who present controlled chronic diseases with no negative impact on physical, psychosocial and mental functionality are considered to be community-dwelling. Psychological and social stress due to unsuccessfully aging can represent a risk for immune system disfunctions. IL-6 levels, isokinetic muscle strength of knee flexion/extension, and functional tests to determine time required to rise from a chair and gait velocity were measured in 57 participants (71.21 +/- 7.38 years). Serum levels of IL-6 were measured in duplicate and were performed within one single assay (mouse monoclonal antibody against IL-6; High-Sensitivity, Quantikine, R & D Systems, USA; intra-assay coefficient of variance = 6.9-7.4%; interassay coefficient of variance = 9.6-6.5%; sensitivity = 0.016-0.110 pg/mL; mean = 0.039 pg/mL). Muscle strength was assessed with the isokinetic dynamometer Biodex System 3 Pro. After the Shapiro-Wilk normality test was applied, correlations were investigated using Spearman and Kruskal-Wallis tests. Post hoc analysis was performed using the Dunn test. A significant negative correlation was observed between plasma IL-6 levels (1.95 +/- 1.77 pg/mL) and muscle strength for knee flexion (70.70 +/- 21.14%; r = -0.265; P = 0.047) and extension (271.84 +/- 67.85%; r = -0.315; P = 0.017). No significant correlation was observed between IL-6 levels and the functional tests (time to rise from a chair = 14.65 +/- 2.82 s and gait velocity = 0.95 +/- 0.14 m/s). These results suggest that IL-6 is associated with reduced muscle strength.

Avaliação das atividades de vida diária de idosos com diferentes níveis de demência / Evaluation of the activities of daily living of elderly people with different levels of dementia

OBJETIVOS: Comparar o desempenho de idosos com diferentes níveis de gravidade de demência em questionários de atividades básicas de vida diária (ABVDs) e atividades instrumentais de vida diária (AIVDs). Verificar se existe correlação entre os questionários de AIVDs aplicados. METODOLOGIA: Foram aleatorizados 90 idosos (75,46 ± 7,66 anos) atendidos no Centro de Referência do Idoso-MG, com diagnóstico clínico de demência (DSM-IV/APA) e classificados quanto ao nível de gravidade da demência (Clinical Dementia Rating). As ABVDs foram avaliadas pelo índice de Katz, e as AIVDs, pelo índice de Lawton-Brody e de Pfeffer. Os testes de Kruskal-Wallis e de Mann-Whitney foram utilizados para verificar o desempenho dos idosos nas ABVDs, e a correlação de Spearman, para investigar a relação entre os instrumentos de AIVDs (alfa< 0,05). RESULTADOS: Foram encontradas diferenças estatisticamente significativas entre o desempenho dos idosos com diferentes níveis de gravidade de demência avaliados pelos instrumentos de ABVDs e AIVDs (p< 0,001). Os questionários de AIVDs apresentaram correlação significativa na amostra total (p< 0,0001; r= -0,818), nos grupos com demência leve (p= 0,007; r= -0,530) e grave (p< 0,0001; r= -0,723). CONCLUSÃO: A gravidade do processo demencial interferiu no desempenho dos idosos nas atividades básicas e instrumentais de vida diária. As AIVDs foram mais comprometidas nos estágios iniciais das demências, e as ABVDs, nos estágios mais avançados. Os questionários de AIVDs utilizados, apesar de sua particularidade estrutural, parecem medir um construto comum. A variabilidade do quadro clínico nos indivíduos com demência moderada pode explicar a ausência de correlação dos questionários usados nesse grupo específico.

OBJECTIVE: To compare the performance of elderly people with different levels of severity of dementia using questionnaires on basic activities of daily living (BADLs) and instrumental activities of daily living (IADLs). Also, to verify whether there were any association between the IADL questionnaires applied. METHOD: Ninety elderly people, aged 75.46 ± 7.66 years with a clinical diagnosis of dementia (DSM-IV/APA) who were seen at the Minas Gerais Reference Center for the Elderly, were randomized selected and classified according to the level of severity of their dementia (Clinical Dementia Rating). Their BADLs were assessed using the Katz Index and their IADLs by the Lawton-Brody and Pfeffer indexes. The Kruskal-Wallis and Mann-Whitney tests were used in order to investigate the elderly people's performance regarding BADLs, while the Spearman correlation was used to investigate the relationships among the IADL (alpha< 0.05). RESULTS: Statistically significant differences in performance were found between the elderly people with different levels of severity of dementia, as assessed by the BADL and IADL questionnaires (p< 0.001). The IADL questionnaires presented significant correlation for the total sample (p< 0.0001; r = -0.818) as well as for the groups with mild dementia (p= 0.007; r = -0.530) and severe dementia (p< 0.0001; r = -0.723). CONCLUSION: The severity of the dementia process interfered with the elderly people's performance of the elderly in BADLs and IADLs. The IADLs were more affected in the early stages of dementia whereas the BADLs were more affected in the more advanced stages. Despite structural particularities, the IADL questionnaires utilized seemed to be measuring a common construct. The variability in the clinical condition of the individuals with moderate dementia may be an explanation for the lack of correlation between the questionnaires in this specific group.

Genetic diversity and antibiotic resistance of clinical and environmental Vibrio Cholerae suggests that many serogroups are reservoirs of resistance.

Vibrio cholerae is an important human pathogen and the cause of cholera. Since genetic variation and antibiotic resistance of strains have implications for effective treatment of the disease, we examined the genetic diversity and antibiotic resistance profile in 92 clinical strains (serogroup O1) and 56 environmental strains (O1 antigen, 42 strains; non-O1 antigen, 14 strains) isolated in Brazil between 1991 and 1999. Clinical and environmental O1 strains showed greater drug resistance compared to environmental non-O1 strains. Nearly all clinical O1 strains were resistant to one or more antibiotics while half of the environmental O1 and non-O1 strains were resistant to one or more antibiotics. No plasmids or class 1 integrons were detected in the strains by PCR analysis. Multilocus enzyme electrophoresis analysis (MLEE) suggests most of the O1 strains belong to a single (South American) clone that is related but different to seventh-pandemic strains isolated from other parts of the world. Our results show that there is a close genetic relationship between clinical and environmental O1 strains and that many serogroups and the environment can be a reservoir for antibiotic resistance.

Transduction of human CD34+ CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors.

The major limitations of Moloney murine leukemia virus (MoMLV)-based vectors for human stem cell applications, particularly those requiring bone marrow (BM) stem cells, include their requirement for mitosis and retroviral receptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third-generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (EGFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopulating cells derived from human BM and cord blood (CB) tested by the SCID-repopulating cell (SRC) assay. Highly purified CD34+ CD38- CB or BM cells were efficiently transduced (4-69%) and stably expressed in EGFP for 40 days in culture following infection for only 24 h without fibronectin, polybrene, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice transplanted with transduced cells from either CB or BM donors were well engrafted, demonstrating maintenance of SRC during the infection procedure. Serially obtained femoral BM samples indicated that the proportion of EGFP+ cells within both myeloid and lymphoid lineages was maintained or even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readily transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropriate for most therapeutic applications, the efficient maintenance and transduction of BM-derived SRC during the short infection procedure are notable advantages of lentivectors.

Expansion of human cord blood CD34(+)CD38(-) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function.

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin(-) CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin(-)CD34(+) CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34(+)CD38(-) cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP(+) human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34(+)CD38(-) phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

Characterization and retroviral transduction of an early human lymphomyeloid precursor assayed in nonswitched long-term culture on murine stroma.

In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Development of a novel osteochondral graft for cartilage repair.

This study reports the development of a novel osteochondral graft for cartilage repair. A technique of proteoglycan extraction via timed enzymatic digestion with hyaluronidase and trypsin and subsequent processing with a chloroform-methanol solution to remove cellular debris from a fresh-frozen bovine osteochondral sample is a method described to prepare a stable biological carrier of low immunogenicity. Lyophilization of the carrier followed by rehydration in a suspension of lapine chondrocytes produced a chimeric xenograft that succeeded in vivo in enhancing cartilage repair. In a pilot study, full-thickness articular cartilage defects treated with these xenografts demonstrated improved healing compared to untreated defects or defects treated with unseeded grafts at 2, 6, and 12 weeks postimplantation. The xenograft provoked a mild inflammatory response; however this did not impede the repair process. Further investigation of this novel chimeric xenograft eventually may yield a method of cartilage repair superior to current methods of treatment.