Longitudinal Associations between Concurrent Changes in Phenotypic Frailty and Lower Urinary Tract Symptoms among Older Men.

BACKGROUND: Lower urinary tract symptoms (LUTS) are associated with prevalent frailty and functional impairment, but longitudinal associations remain unexplored. OBJECTIVES: To assess the association of change in phenotypic frailty with concurrent worsening LUTS severity among older men without clinically significant LUTS at baseline. DESIGN: Multicenter, prospective cohort study. SETTING: Population-based. PARTICIPANTS: Participants included community-dwelling men age ≥65 years at enrollment in the Osteoporotic Fractures in Men study. MEASUREMENTS: Data were collected at 4 visits over 7 years. Phenotypic frailty score (range: 0-5) was defined at each visit using adapted Fried criterion and men were categorized at baseline as robust (0), pre-frail (1-2), or frail (3-5). Within-person change in frailty was calculated at each visit as the absolute difference in number of criteria met compared to baseline. LUTS severity was defined using the American Urologic Association Symptom Index (AUASI; range: 0-35) and men with AUASI ≥8 at baseline were excluded. Linear mixed effects models were adjusted for demographics, health-behaviors, and comorbidities to quantify the association between within-person change in frailty and AUASI. RESULTS: Among 3235 men included in analysis, 48% were robust, 45% were pre-frail, and 7% were frail. Whereas baseline frailty status was not associated with change in LUTS severity, within-person increases in frailty were associated with greater LUTS severity (quadratic P<0.001). Among robust men at baseline, mean predicted AUASI during follow-up was 4.2 (95% CI 3.9, 4.5) among those meeting 0 frailty criteria, 4.6 (95% CI 4.3, 4.9) among those meeting 1 criterion increasing non-linearly to 11.2 (95% CI 9.8, 12.6) among those meeting 5 criteria. CONCLUSIONS: Greater phenotypic frailty was associated with non-linear increases in LUTS severity in older men over time, independent of age and comorbidities. Results suggest LUTS and frailty share an underlying mechanism that is not targeted by existing LUTS interventions.

Comparison of treatment outcomes with vancomycin alone versus combination therapy in severe Clostridium difficile infection.

BACKGROUND: The recommended treatment for severe Clostridium difficile infection (CDI) is oral vancomycin alone. Combination therapy with metronidazole is only recommended in cases complicated by shock, ileus, or toxic megacolon. However, patients with severe infection are often treated with combination therapy despite a lack of data supporting this practice. AIM: To evaluate differences in outcomes for patients with severe CDI treated with oral vancomycin alone versus combination therapy. METHODS: Medical records of 78 patients with severe CDI receiving either oral vancomycin alone or combination therapy for ≥ 72h were retrospectively reviewed. The primary outcome was time to clinical cure of CDI, defined as the first day of resolution of diarrhoea for ≥ 48h without development of a complication. Other endpoints included cure rates, complication rates, and recurrence rates. FINDINGS: There was no difference in the incidence of clinical cure between monotherapy and combination therapy (57.1% vs 65.1%, P = 0.49). Median time to clinical cure was 7.0 days for the monotherapy group and 8.0 days for combination therapy (P = 0.19). After adjustment for potential confounders, the hazard ratio of the time to clinical cure for combination therapy compared with monotherapy was 0.58 (P = 0.10). There was no difference in recurrence rate or rates of individual complications between groups; however, there was a significantly higher composite complication rate in the combination therapy group. CONCLUSION: These data suggest that there is no difference in treatment outcomes between monotherapy and combination therapy for severe CDI.

Localization of recombination activating gene 1/green fluorescent protein (RAG1/GFP) expression in secondary lymphoid organs after immunization with T-dependent antigens in rag1/gfp knockin mice.

Secondary rearrangements of immunoglobulin gene segments that generate a new antibody repertoire in peripheral B cells have been described as receptor revision and occur by as yet unknown mechanisms. To determine the importance of recombination activating gene (RAG) expression in receptor revision, heterozygous rag1/green fluorescent protein (gfp) knockin mice were used to examine the location of RAG1 expression in the germinal centers (GCs) of lymphoid follicles after immunization with a variety of T-cell-dependent antigens. Immunization of rag1/gfp heterozygous mice or rag1 homozygous knockout mice reconstituted with rag1/gfp heterozygous spleen cells caused the down-regulation of RAG1/GFP signal in GCs. Although some RAG1/GFP(+) cells appeared in regions surrounding the peanut agglutinin (PNA)(+)GL-7(+) GC area, RAG1/GFP(+) cells did not accumulate in the central region. In addition, the stimulation of spleen B cells with anti-mu antibody plus interleukin-4 (IL-4) or with anti-CD40 monoclonal antibody plus IL-7 did not induce GFP signals at detectable levels in vitro. These results clearly demonstrate that RAG1 re-expression either does not occur or is at extremely low levels in antigen-driven B cells in GCs of secondary lymphoid follicles, suggesting that other mechanisms may mediate the gene rearrangements observed in receptor revision.

Interaction of abl and raf with IL-7 signaling pathway and transformation of pre-B cells from resistant mice.

After in vivo inoculation with abl/myc- and raf/myc-containing retroviruses, BALB/c mice predominantly develop late stage B cell tumors (plasmacytomas) and less frequently develop earlier B-lineage tumors while DBA/2 mice do not develop B-lineage tumors. We have investigated the in vitro tumorigenic potential of these viruses using cultured normal pre-B cell lymphocytes from both BALB/c and DBA/2 mice. Interestingly, both viruses infect cultured pre-B lymphocytes from both mouse strains. Following infection, IL-7 dependent pre-B cells become independent of normal in vitro growth requirements within 24 h and can rapidly form in vivo pre-B lymphomas in both mouse strains. Mechanisms mediating loss of IL-7 dependence are different depending on whether the raf or abl gene is present in myc-containing viruses. IL-7 JAK-STAT signaling is constitutively active in abl/myc induced pre-B cell tumors. In contrast, IL-7 JAK-STAT signaling is not constitutive in raf/myc induced pre-B cell tumors, demonstrating that subversion of this component of IL-7 signal transduction is not obligatory for pre-B cell transformation or loss of IL-7 dependence.

Modulated expression of the epidermal growth factor-like homeotic protein dlk influences stromal-cell-pre-B-cell interactions, stromal cell adipogenesis, and pre-B-cell interleukin-7 requirements.

A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.

A role for c-myc in the regulation of thymocyte differentiation and possibly positive selection.

The purpose of thymocyte differentiation is to establish the T cell repertoire and eliminate nonfunctional and autoreactive T cells. In an analysis of potential regulators of this process, we have found that c-myc expression is down-regulated dramatically during early thymocyte differentiation and subsequently up-regulated along with TCR/CD3 in CD4+8+ cells. Transgenic E beta-myc mice that constitutively express c-myc in thymocytes have a larger proportion of thymocytes with high TCR/CD3 and low heat-stable antigen-1 expression than controls, and an increase in the number of transitional cells with a CD4+CD8low phenotype. Mature E beta-myc T cells respond less vigorously than controls to activation through their TCR/CD3 complex, as measured by proliferation and induction of the activation marker CD69. These results are consistent with a hypothesis in which activation of immature T cells through TCR/CD3 induces c-myc up-regulation and drives thymocytes through the initial stage of positive selection.

Transforming growth factor-beta 1 null mice. An animal model for inflammatory disorders.

Approximately 40% of transforming growth factor-beta 1 null (knockout) mice generated in our laboratory develop normally to term, but 60% die in utero. The animals appear normal during the first 2 weeks of life but develop a rapid wasting syndrome and die by 3 to 4 weeks of age. All of the knockout mice have a multifocal inflammatory disease in many tissues. The heart and lungs are most severely affected. Increased adhesion of leukocytes to the endothelium of pulmonary veins is the initial lesion seen at day 8 postnatally and is soon followed by perivascular cuffing as well as inflammatory infiltrates in lung parenchyma. The lesions in the heart begin as endocarditis and then progress to myocarditis and pericarditis. Within the lung, chronic inflammatory infiltrates consist of T and B lymphocytes, including plasma cells, whereas macrophages are the primary inflammatory cell type in the heart. Increased expression of major histocompatibility complex class I and II proteins is seen in pulmonary vascular endothelium as early as day 8. An immunoblastic response in mediastinal and mandibular lymph nodes and spleen is also seen. In the absence of any pathogens, this massive inflammatory disease, together with overexpression of major histocompatibility complex class I and II proteins and overproduction of immunoglobulins by lymphocytes, offers circumstantial evidence for an autoimmune etiology.

Loss of p53 expression in Myc-induced B lineage tumors.

Tumors are formed following the accumulation of several genetic changes in genes which normally function to regulate cell growth. As yet it is unclear why multiple mutations are required, which type of alterations can collaborate with each other, and if collaboration is cell-type specific. In our myc transgenic mouse model system both point mutations and loss of mRNA expression for the p53 tumor suppressor gene have been found in the myc-induced B-lineage tumors arising spontaneously in these mice. This demonstrates the collaboration between these two growth control genes in cellular transformation. The observation that alterations in the expression of p53 is a common phenomenon in tumors formed in myc transgenic mice as well as a variety of different types of human tumors suggests that inactivation of the p53 growth control pathway may be required for transformation, and that alterations in p53 itself might be the most efficient way to achieve this inactivation. An analysis of the molecular mechanism for p53 alterations has implications for what kind of factors, both environmental and physiological, can influence tumor formation. The identification of collaboration groups has implications for the process of tumor formation, growth regulation, and will some day be important for the diagnosis of cancer, the prognosis of the individual and the design of specific therapeutic agents for treatment.

Pre-B lymphocyte-specific transcriptional control of the mouse VpreB gene.

The VpreB genes, which encode surrogate immunoglobulin light chain molecules, are expressed as RNA almost exclusively in pre-B cells. We have investigated the transcriptional control mechanisms which are responsible for the pre-B cell-specific RNA expression of the mouse VpreB1 and VpreB2 genes. Nuclear run-on analyses demonstrate that the pre-B cell-specific expression of both VpreB genes is controlled primarily at the level of initiation of transcription. S1 nuclease protection-mapping defined two or three major start sites of transcription for the VpreB genes. To find a promoter and other potential cis-acting regulatory elements, a 700-bp fragment 5' of the transcription start sites of the VpreB1 gene was used in gene transfer experiments and found to act as a promoter in pre-B lymphocytes. Deletion experiments showed that 191 bp upstream of the most 5' transcription start site is required for the pre-B cell promoter activity. DNA sequence analysis of the 5' region of the mouse VpreB1, VpreB2 and human VpreB genes reveal that this region of approximately 200 bp is strongly conserved. This 200-bp promoter region contains several conserved nucleotide sequence motifs which may act to mediate the pre-B cell-specific transcription of the VpreB genes.

VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias.

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.

Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas.

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Restriction fragment analysis of V preB and lambda 5 within the genus Mus.

DNA from a panel of inbred strains of mice and colony bred mice, isolated from different geographical locations, was hybridized to mouse V preB and lambda 5 probes under stringent conditions, indicating sequence similarities greater than 80%. The probe for lambda 5 detects one gene and the probe for V preB detects two genes (V preB1 and V preB2) in the inbred strains of mice examined under the stringency used. No restriction endonuclease fragment length polymorphisms (RFLP) were detected with the V preB and lambda 5 DNA probes among the inbred strains of mice using Bam HI and Hind III. Very few RFLP were detected among Mus musculus subspecies, and the intensity of the hybridization did not differ significantly with either DNA probe. The number of RFLP increased slightly when different species and subgenera were examined, and the intensity of the hybridization signal began to decrease in samples from the different subgenera, suggesting a slight decrease in sequence similarity for both V preB genes with increased time of divergence. Fewer RFLP were detected with the lambda 5 DNA probe. DNA from 11 different Mus species representing 4 subgenera, genetically isolated from laboratory mice for approximately 1-12 million years, continued to hybridize under high stringency conditions using both DNA probes. A comigrating lambda 5 and V preB restriction endonuclease fragment was detected in most of the samples examined, suggesting the close physical linkage of V preB1 and lambda 5 is maintained within the genus Mus. These results suggest that V preB1, V preB2 and lambda 5 have been present for over 12 million years.

Cellular stages and molecular steps of murine B-cell development.

Development of B cells in fetal liver occurs in one synchronous wave and involves probably no more than four critical divisions. This leads us to suggest that the main pool of proliferating progenitors that replenish the peripheral B-cell pool are progenitors before Ig gene rearrangement, that the four Ig gene rearrangements (DH to JH, VH to DHJH, VK to JK, and V lambda to J lambda) might occur in four critical divisions, and that a stromal-cell-dependent phase of pre-B development in which all rearrangements are made is succeeded by a stromal-cell-independent phase of sIG+ pre-B-cell maturation to mature mitogen-reactive B cells. We speculate on the molecular nature of the tightly controlled steps of Ig rearrangements during pre-B-cell development that might involve the pre-B-cell specific genes Vpre-B and lambda 5 and the B-lineage-specific gene mb-1 in interactions with stromal cells.

Structure and pre-B lymphocyte restricted expression of the VpreB in humans and conservation of its structure in other mammalian species.

DNA from several mammals, including humans, was found to contain one or more restriction enzyme digested DNA fragments which hybridized to the mouse VpreB gene under stringencies demonstrating at least 70% nucleotide sequence homologies, indicating that the VpreB locus may be widespread and highly conserved among mammals. A human VpreB genomic clone was isolated and sequenced. Two exons and the intervening intron are spaced almost identically as in the mouse VpreB1 gene, and show 76% sequence homology to the mouse gene. As in the mouse VpreB1 gene, the 5' end of the human VpreB gene contains characteristic features of Ig domains, while the 3' end is Ig non-related. This 3' Ig non-related structure of the VpreB gene(s) may, therefore, have existed before the speciation of humans and mice over 65 million years ago. Sequences encoding the entire putative second framework region and a stretch in the third framework region are identical in human and mouse VpreB. the human VpreB gene appears to be selectively expressed in human pre-B cell lines as an 0.85 kb poly(A)+ RNA. Its expression promises to be a useful marker for the detection of normal and malignant human pre-B lymphocytes.