Implementation of measurement instruments in physical therapist practice: development of a tailored strategy.

BACKGROUND AND PURPOSE: The use of measurement instruments has become a major issue in physical therapy, but their use in daily practice is infrequent. The aims of this case report were to develop and evaluate a plan for the systematic implementation of 2 measurement instruments frequently recommended in Dutch physical therapy clinical guidelines: the Patient-Specific Complaints instrument and the Six-Minute Walk Test. CASE DESCRIPTION: A systematic implementation plan was used, starting with a problem analysis of aspects of physical therapist practice. A literary search, structured interviews, and sounding board meetings were used to identify barriers and facilitators. Based on these factors, various strategies were developed through the use of a planning model for the process of change. OUTCOMES: Barriers and facilitators were revealed in various domains: physical therapists' competence and attitude (knowledge and resistance to change), organization (policy), patients (different expectations), and measurement instruments (feasibility). The strategies developed were adjustment of the measurement instruments, a self-analysis list, and an education module. Pilot testing and evaluation of the implementation plan were undertaken. The strategies developed were applicable to physical therapist practice. Self-analysis, education, and attention to the practice organization made the physical therapists aware of their actual behavior, increased their knowledge, and improved their attitudes toward and their use of measurement instruments. DISCUSSION: The use of a planning model made it possible to tailor multifaceted strategies toward various domains and phases of behavioral change. The strategies will be further developed in programs of the Royal Dutch Society for Physical Therapy. Future studies should examine the use of measurement instruments as an integrated part of the process of clinical reasoning. The focus of future studies should be directed not only toward physical therapists but also toward the practice organization and professional associations.

Mutagenesis of a cAMP response element within the latency-associated transcript promoter of HSV-1 reduces adrenergic reactivation.

Mutagenesis of a cyclic AMP response element (CRE) within the LAT promoter of HSV-1 reduces the ability of LAT expression to be induced in transient assays, but has only a minimal impact on reactivation of the virus in in vitro systems. Here we show that a CRE mutation results in a significant reduction of adrenergically induced reactivation in vivo in the rabbit eye model. Spontaneous reactivation frequencies were also reduced. In addition, we demonstrate that this mutation has no effect on the amount of LAT expressed during latency when compared with the parent, 17syn+, and the rescuant. These results indicate a greater effect of CRE on induced reactivation in vivo than in in vitro systems, but also suggest that the CRE in the LAT promoter is not autonomous in conducting the reactivation signal.

Murine cytomegalovirus DNA in peripheral blood of latently infected mice is detectable only in monocytes and polymorphonuclear leukocytes.

Cytomegalovirus (CMV), as do other herpesviruses, establishes a lifelong latent infection in its natural host. While in immunologically intact hosts most CMV infections are subclinical, clinical disease follows severe immunosuppression and immunodeficiency. In these situations CMV may produce serious life-threatening disease, and virus reactivated from the latent state is often responsible. Essential to understanding this virus and its pathogenesis is the need to define particular tissue and cell types harboring viral DNA. We searched for viral DNA and RNA in subpopulations of blood cells from mice latently infected with murine CMV by using differential centrifugation and fluorescent antibody cell sorting followed by polymerase chain reaction analysis. Following intravenous inoculation, the viral DNA was found to be present in the buffy coat at and after 21 days postinfection, and both granulocytes and peripheral blood mononuclear leukocytes (PBML) were reservoirs. Further analysis of the PBML fraction by separation into Mac-1+ and Mac-1- cells revealed that monocytes harbored the DNA while lymphocytes were not sites of persistence. We conclude that in buffy coat of latently infected mice the viral DNA is present only in cells of the myeloid lineage. The relationship of this DNA to the latent infection is discussed.

Long-term expression of a foreign gene from a unique position in the latent herpes simplex virus genome.

As a result of its capacity to establish and maintain a life-long latent infection in the nervous system, herpes simplex virus (HSV) has been promoted as an ideal vector for introducing DNA into mature, differentiated, post-mitotic neurons. Although delivery of foreign genes into neurons using HSV vectors has been well established, the potential of these vectors for scientific inquiry or therapeutic use has been hampered by the lack of efficient long-term expression of these foreign genes. In the few instances where expression from the latent genome has been reported, expression appears to be minimal and levels of mRNA present have not been established. Here we describe HSV viral vectors that express a foreign gene during latency in dorsal root ganglia (DRG). More particularly, we have constructed a vector that, by histochemical assays for the protein, expresses the beta-galactosidase (beta-Gal) gene for at least 18 months post infection. We have further characterized the expression of beta-Gal transcripts by quantitative reverse transcription polymerase chain reaction (RT-PCR) and determined that there are 32,000 copies of beta-Gal transcripts per 0.5 microgram of total RNA at 18 months post infection. The vector makes use of the mouse Moloney leukemia virus (MMLV) long terminal repeat (LTR) promoter located directly upstream from the latency-associated transcripts (LAT) promoter region and expresses mRNA from the DNA strand opposite to that expressing the LAT. Finally, the vector was constructed using a system that allows other promoter/gene constructs to be easily inserted into the viral genome. It may have utility in studying the effects of cellular or viral gene expression on establishment, maintenance or reactivation from latency or for the delivery and expression of therapeutic proteins employed in gene therapy of the nervous system.

Expression of the lacZ reporter gene in the rat basal forebrain, hippocampus, and nigrostriatal pathway using a nonreplicating herpes simplex vector.

We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy.

A 348-base-pair region in the latency-associated transcript facilitates herpes simplex virus type 1 reactivation.

Latency-associated transcript (LAT) promoter deletion mutants of herpes simplex virus type 1 have a reduced capacity to reactivate following adrenergic induction in the rabbit eye model. We have mapped a reactivation phenotype within LAT and describe the construction of recombinants in which poly(A) addition sites have been placed at intervals within the LAT region to form truncated LAT transcripts. These mutants localize the induced reactivation phenotype to the 5' end of LAT. To further define this region, we constructed a recombinant containing a 348-bp deletion located 217 bp downstream of the transcription start site of the 8.5-kb LAT. This virus, 17delta348, expresses LAT but exhibits a significantly reduced ability to reactivate following epinephrine iontophoresis into the cornea. Quantitative DNA PCR analysis reveals that 17delta 348 establishes a latent infection within rabbit trigeminal ganglia with the same efficiency as does either the rescuant or wild-type virus. The region deleted in 17delta348 encodes three potential translational initiators (ATGs) which we have mutated and demonstrated to be dispensable for epinephrine-induced reactivation. In addition, three smaller deletions within this region have been constructed and were shown to reactivate at wild-type (parent) frequencies. These studies indicate that an undefined portion of the 348-bp region is required to facilitate induced reactivation. Sequence analysis of this 348-bp region revealed a CpG island which extends into the LAT promoter and which possesses homology to conserved elements within the mouse and human XIST transcript encoded on the X chromosome. Possible implications of these elements in the regulation of LAT expression are discussed.

Neuroinvasive properties of herpes simplex virus type 1 glycoprotein variants are controlled by the immune response.

Neuroinvasiveness is a property central to the pathogenesis of herpes simplex virus (HSV) and most isolates demonstrate this property. Exceptions are HSV strains KOS and ANG, for which we have previously shown that the non-neuroinvasive phenotype is referable to single amino acid changes in glycoprotein D for ANG or glycoprotein B for KOS. Because glycoproteins B and D are immunologically significant, the possibility that the phenotype has an immunologic basis was examined. Nonimmunosuppressed mice could not be killed with any dose of these non-neuroinvasive viruses after footpad inoculation, but in cyclophosphamide-suppressed animals, the ratios of plaque-forming units to LD50 decreased by at least four orders of magnitude to levels comparable with that of ANG-path, a neuroinvasive derivative of ANG. KOS and ANG induced a more rapid circulating neutralizing Ab response than did ANG-path, and mice were protected when these agents were co-infected with the neuroinvasive strain. The noninvasive viruses engendered an enhanced mononuclear cell infiltrate in infected spinal ganglia which consisted of increased numbers of CD4+ and CD8+ T cells and an increased production and secretion of IgG. HSV-specific Ab-secreting cells were also observed. In addition, passive transfer of anti-HSV mouse serum protected immunosuppressed mice from lethal HSV challenge. Selective in vivo depletion of T lymphocytes increased the detectable levels of both KOS and ANG viruses in the spinal ganglia at 6 days postinfection, but it did not alter the ratios of plaque-forming units to LD50 or affect the HSV-induced increase in ganglionic IgG. Taken together, these data indicate that in these systems there is an immunologic basis for the control of HSV-1 neuroinvasiveness and that humoral, rather than cell-mediated immunity, is playing the major role.

Long-term expression of a reporter gene from latent herpes simplex virus in the rat hippocampus.

A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.

Murine cytomegalovirus is present in both chronic active and latent states in persistently infected mice.

Cytomegalovirus induces serious disease in immunosuppressed individuals, often from an "activated" persistent infection. Whether the infection is chronically active or latent is unknown. Using murine cytomegalovirus (MCMV) in mice as a model system, we examined persistent infections in spleen, lung, and bone marrow of infected animals. At 28 days after infection, no virus could be recovered from any organs tested except salivary glands, and here, virus was cleared by 48 days. Virus could be retrieved at all times by cocultivation of spleen or lung with permissive cells. In addition, MCMV DNA was always present in spleen, lung, and bone marrow. After acute infection, RNA from the MCMV immediate early-1 (ie-1) gene was routinely found only in the lung. In spleen and bone marrow, only one sample from each organ examined at these times contained ie-1 RNA, and the RNA in these two samples was present at levels comparable to that found in acute infection. This suggests that the virus had reactivated. The ie-1 RNA found in the lung was present at a much lower RNA:DNA ratio than that found at early times. Taken together, these results indicate that persistent MCMV exists simultaneously in both chronic active and latent states.

Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene.

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.

Herpes simplex virus type 1 DNA replication and gene expression during explant-induced reactivation of latently infected murine sensory ganglia.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus DNA replication and gene expression in two murine in vitro models for virus reactivation. We examined latent infections with wild-type (wt), precisely defined latency-associated transcript-negative (LAT-) mutants, and LAT+ rescuants of these mutants of the 17syn+ strain of virus in both murine trigeminal and lumbosacral ganglia and of the KOS(M) strain in the latter. In explants of ganglia latently infected with the LAT- mutant of strain 17syn+ virus, a reduction in number of cultures exhibiting cytopathic effects due to virus reactivation and measurable delays in virus recovery were observed compared with wt or the LAT+ rescuant. This LAT-specific effect was not seen in explants of lumbosacral ganglia latently infected with mutants derived from the KOS(M) strain of virus. Although there was appreciable variation between individual animals, no significant difference between LAT+ and LAT- virus in time of onset of viral DNA replication in explanted ganglia was seen with use of either virus strain. There was a slight decrease in the relative amount of viral DNA recovered compared with internal cellular controls in latently infected ganglia harboring the LAT- mutant of 17syn+ compared with the wt virus or the LAT+ rescuant. This reduced relative amount ranged from 0 to as much as 50% but averaged 20%. Such differences were not seen in infections with KOS(M)-derived mutants. In contrast, although expression of productive-cycle transcripts could be detected within 4 h following explant cultivation of latently infected ganglia, no differences between LAT+ and LAT- viruses could be seen. As discussed, these data place specific constraints on possible models for the role of LAT expression in in vitro reactivation systems.

Molecular analysis of herpes simplex virus type 1 during epinephrine-induced reactivation of latently infected rabbits in vivo.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus (HSV) DNA replication and gene expression in the rabbit corneal model for virus reactivation in vivo. We used carefully defined latency-associated transcript-negative (LAT-) and LAT+ promoter mutants of the 17syn+ strain of HSV type 1. In agreement with earlier studies using a more extensive LAT- deletion mutant, the 17 delta Pst(LAT-) virus reactivated with extremely low frequency upon epinephrine induction. In contrast to our findings with murine latency models, amounts of viral DNA recovered from rabbit ganglia latently infected with either LAT+ or LAT- virus were equivalent. Also in contrast with the murine models, no net increase in viral DNA was seen in latently infected rabbit trigeminal ganglia induced to reactivate in vivo by iontophoresis of epinephrine. Despite this, transcription of lytic-phase genes could be detected within 4 h following induction of rabbits latently infected with either LAT+ or LAT- virus; this transcription diminished by 16 h following induction. These results are discussed in relation to models for the mechanism of action of HSV LAT.

Decreased reporter gene expression during latent infection with HSV LAT promoter constructs.

The latency-associated transcripts (LAT), which code from an 8.5 kb segment of the internal repeat region of the HSV genome, are the only viral transcripts that are present during HSV latent infection. However, little is known about the relative contribution of promoter activity, degradative processes, and elements or regions affecting long term expression of these transcripts in latently infected neurons. To begin to address this question we investigated LAT promoter activity during acute and latent infection. Mouse footpads were infected with KOS/62-3, an engineered herpes simplex virus in which both copies of the LAT promoter are used to drive expression of the Escherichia coli lac Z gene. Four days post-inoculation (p.i.) abundant beta-galactosidase (beta-gal) protein and transcripts were present within ganglionic neurons as assayed by enzyme histochemistry and in situ hybridization. In contrast, by Day 21 (at which time a latent infection had been established) no beta-gal transcripts were present in infected ganglia, even when assayed by the polymerase chain reaction (PCR). These findings indicate a significant drop in LAT promoter activity between Day 4 and Day 21 p.i. To provide confirmatory evidence for this conclusion we infected mice with a second viral construct, KOS/67-7, in which the LAT promoter was used to drive expression of the nerve growth factor (NGF) gene. Four days p.i., abundant NGF antigen and transcripts were present in infected ganglionic neurons, but no evidence of transcription of the cloned NGF gene could be found in latently infected ganglia. Our findings suggest that LAT promoter activity is severely restricted during the latent phase of ganglionic infection.

Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness.

Herpes simplex virus type 1 strains ANG and KOS lack neuroinvasiveness when inoculated on the footpads of mice, and because the strains are able to complement each other, the genes associated with this phenotype differ. In this study, we used marker rescue techniques to show that at least two genes cloned from ANG are required to restore neuroinvasiveness to KOS. One of the two fragments required is the 6.3-kb BamHI-A/EcoRI-D fragment (0.15 to 0.19 map units). The second has been identified as the sequence encoding glycoprotein B (gB) (UL27). Analysis of ANG and KOS DNA sequences in the relevant region of the gB gene revealed two nucleotide differences which result in amino acid differences in the gB protein. One appears to be unique to the strain of KOS used in our laboratory. The second, at codon 523 of the mature gB protein, encodes a valine in KOS and an alanine in ANG. Recombinant KOS viruses which contained ANG sequences in this region were constructed, and two independently selected recombinants demonstrated increased neuroinvasiveness in mice. From these results, we conclude that gB significantly influences neuroinvasiveness. Mechanisms by which this might occur are discussed.

The herpes simplex virus type 1 reactivation function lies outside the latency-associated transcript open reading frame ORF-2.

The latency-associated transcription unit has been shown to be important for in vivo reactivation of herpes simplex virus from the latent state. A recombinant virus was constructed to alter the largest open reading frame in this region. This virus had a wild-type reactivation phenotype, suggesting that herpes simplex virus does not require a protein function from this reading frame for efficient reactivation from latency.

Latent infection can be established with drastically restricted transcription and replication of the HSV-1 genome.

Viral functions essential for the establishment of latent infection in murine sensory neurons in vivo were investigated by employing a herpes simplex virus type 1 (HSV-1) variant (KD6/B11) deleted for expression of the ICP4 gene and therefore unable to replicate. Since the viral DNA persisted in these cells, the latency-associated transcripts were expressed for prolonged periods of time, and the variant was biologically retrievable by superinfection with an ICP4-competent agent, we concluded that a latent infection had been established. In situ hybridization experiments designed to investigate gene expression during the acute phase of infection with the variant revealed a highly restricted pattern compared to that of the wild-type parent virus HSV-1 KOS(M). While latency-associated transcripts were detected in a large number of infected neurons, expression of other virus genes was limited to a subset of immediate-early and early genes (ICP0, ICP8, ICP27, and HSV-1 DNA polymerase genes). Expression was further limited to a small proportion of the infected neurons (approximately 1% of neurons expressing latency-associated transcripts). No hybridization was detected with probes specific for the viral TK gene and late genes VP5 and gC. Quantitative assays of viral DNA during the acute phase of infection indicated that the input viral DNA did not replicate. From these results we conclude that HSV-1 latent infection can be established in murine sensory neurons under conditions in which viral genetic expression and DNA replication are severely restricted.

HSV-1 neuroinvasiveness.

Neuroinvasiveness, the capacity of virus to enter and progress through the nervous system, may be accomplished by hematogenous or neural routes. We have been interested in defining HSV-1 genes specifically concerned with the neural pathway. In one system (involving HSV strain Ang), we have found that a single amino acid change in glycoprotein D, a viral membrane protein, confers invasiveness upon a noninvasive agent. Preliminary studies of another noninvasive agent, KOS, suggests that at least 2 genes are related to the phenotype. Experiments which establish specificity for the noninvasive phenotype and a discussion of the potential mechanisms involved in the glycoprotein D localization are also discussed.

Pathways of viral gene expression during acute neuronal infection with HSV-1.

Pathways of viral gene expression were investigated during the acute phase of sensory ganglionic infection with HSV-1. To facilitate these studies we constructed KOS/62-3, an HSV-1 vector in which the Escherichia coli lac-Z gene was inserted behind both copies of the promoter for the viral latency-associated transcripts. Following footpad inoculation of mice with the virus, acutely infected dorsal root ganglion (DRG) neurons were assayed by dual immunofluorescence for the presence of beta-galactosidase and HSV viral antigens. Most infected neurons stained for either beta-galactosidase or viral antigens. Less than 0.2% of neurons staining for viral antigens also expressed beta-galactosidase, and less than 10% of neurons expressing beta-galactosidase also stained for viral antigen. As a consequence of these findings, we propose that there are essentially two populations of HSV-infected neurons during the acute phase of ganglionic infection. In one population of neurons there is abundant viral protein synthesis but minimal transcription of latency-associated transcripts, whereas in a second population of neurons viral gene expression is severely restricted except for the synthesis of latency-associated transcripts. Since DRG neurons are a heterogeneous population of cells, we further sought to determine whether either pathway of gene expression was more likely to occur in a particular neuronal phenotype. To accomplish this, antibodies were used to characterize the DRG neuronal phenotypes acutely infected with the virus. The results indicated that the pathway of neuronal infection characterized by transcription of abundant latency-associated transcripts and minimal viral protein synthesis was much more likely to occur in DRG neurons expressing the cellular antigen SSEA-3. These data indicate that the neuron plays a major role in regulating the outcome of infection with HSV. Finally, we sought to determine whether DNA replication occurs in the course of establishment of a latent infection. We found that the DNA content of neurons latently infected with KOS(M) strain HSV was not affected by treatment with nucleotide analogues during the acute phase of ganglionic infection, suggesting that viral DNA replication does not occur during the establishment of latent infection.

Differential accumulation of herpes simplex virus type 1 latency-associated transcripts in sensory and autonomic ganglia.

We have analyzed the capacity of sensory and autonomic ganglia to demonstrate latency-associated transcripts (LATs) following inoculation of the anterior chamber of the mouse eye with Herpes simplex virus type 1 (HSV-1). In autonomic ganglia, the number of LAT-containing neurons decreased 50-fold or more from the acute to the latent phase, while in the trigeminal ganglion, the decrease was less than 2-fold. The decrease in autonomic ganglia could not be related to destruction of neurons expressing LATs, since these ganglia harbored substantial amounts of viral DNA. The data demonstrate that during the latent phase of the infection, accumulation of LATs varies depending on the type of infected neuron and suggest that some neurons may harbor a latent infection in the absence of LAT expression.