Iatrogenic systemic lymphedema following multiple myeloma treatment.

Lymphedema is a debilitating disease characterized by abnormal lymphatic drainage, either due to primary maldevelopment of the lymphatic system or to secondary injury. The clinical features of primary and secondary lymphedema differ, with primary lymphedema more often involving progressive bilateral lower extremity disease as compared to secondary lymphedema characteristically having more localized symptoms related to the origin of injury. This case presentation describes a patient who presented with bilateral lower extremity swelling, left greater than the right, with imaging results to support the diagnosis of lymphedema. During the time he was followed in our clinic, our team witnessed rapid progression of his lymphedema despite compliance with conservative management. We believe that the primary mechanism of systemic damage to our patient's lymphatic system is the lenalidomide and bortezomib therapy prescribed to treat multiple myeloma. This review explores the relationship between lenalidomide, bortezomib, and lymphedema in efforts of understanding this unique pathology of iatrogenic lymphedema mimicking primary nature.

Reconciliation and Disposal of Oral Medication: Creating a Safe Process for Clinical Research Personnel.

Clinical research nurses and non-licensed study coordinators observed variation in procedures for reconciliation and disposal of oral investigational medications across the institution. An academic medical center implemented a quality improvement project to standardize the process of reconciliation and disposal of oral investigational medications. An interprofessional collaborative workgroup was formed, which led to multiple interventions to standardize practice, including revision of three policies and procedures, redesign of specific work areas to establish drug-counting rooms, review of personal protective equipment requirements, revision of educational training, and regular cleaning of potentially contaminated workstations.

Wide Variability in Prethrombectomy Workflow Practices in the United States: A Multicenter Survey.

BACKGROUND AND PURPOSE: Clinical outcomes in patients with acute ischemic stroke caused by large vessel occlusion depend on the speed and quality of workflows leading to mechanical thrombectomy. In the absence of universally accepted best practices for workflow, developing stroke hospitals can benefit from improved awareness of real-world workflows in effect at experienced centers. To this end, we surveyed prethrombectomy workflow practices at stroke centers throughout the United States. MATERIALS AND METHODS: E-mail and phone interviews were conducted with neurointerventional team members at 30 experienced, endovascular-capable stroke centers. Questions were chosen to reflect workflow components of triage, team activation, transport, case setup, and anesthesia. RESULTS: There is wide variation in prethrombectomy workflows. At 53% of institutions, nonphysician staff respond to stroke alerts alongside physicians. Imaging triage involves noninvasive angiography or perfusion imaging at 97% and 63% of institutions, respectively. Neurointerventional consultation is initiated before the completion of neuroimaging at 86% of institutions, and the team is activated before a final treatment decision at 59%. The neurointerventional team most commonly arrives within 30 minutes. Patients may be transported to the neuroangiography suite before team arrival at 43% of institutions. Procedural trays are set up in advance of team arrival at 13% of centers; additional thrombectomy devices are centrally stored at 54%. A power injector for angiographic runs is consistently used at 43% of institutions. Anesthesiology routinely supports thrombectomies at 67% of institutions. CONCLUSIONS: Prethrombectomy workflows vary widely between experienced centers. Improved awareness of real-world workflows and their variations may help to guide institutions in designing their own protocols of care.

Chronic GvHD-associated serositis and pericarditis.

Serositis is a rare manifestation of chronic GvHD (cGvHD). No risk factors or laboratory changes associated with this syndrome have been recognized to date, and outcomes have not been described in a large series. We searched our institutional database for patients undergoing allogeneic hematopoietic cell transplant identified as having serositis or pericarditis. Laboratory studies from prior to diagnosis, at diagnosis and post diagnosis of serositis, as well as outcomes from invasive procedures were included. Twenty patients met criteria for cGvHD-associated serositis, and all but three patients had a prior diagnosis of cGvHD. Fifteen were male, and the complication occurred in the setting of immunosuppressant taper in 12 cases. Ten patients required invasive interventions, including pericardial window or stripping. A significant increase in blood monocytes and decrease in serum albumin were identified at diagnosis compared with pre-diagnosis. Out of 20 patients, 17 were treated with steroids, with 12 demonstrating a complete response. These data suggest that cGvHD-associated serositis occurs mainly in the setting of treated as opposed to de novo cGvHD and biomarkers associated with the syndrome include a decrease in albumin and an increase in absolute monocyte count. Outcome data from larger series are required to better understand the optimal management of this rare complication.

Cross-sectional validation study of patient-reported outcomes in patients with paroxysmal nocturnal haemoglobinuria.

BACKGROUND: Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired, clonal haemopoietic stem cell disorder that causes chronic intravascular haemolysis, increases the risk of thrombosis and results in significant patient morbidity and mortality. The symptoms of PNH may have a major impact on patient quality of life. AIMS: To assess patient fatigue and health-related quality of life in 29 patients with PNH using the Functional Assessment of Chronic Illness Therapy Fatigue subscale version 4 (FACIT-Fatigue) and the European Organization for Research and Treatment of Cancer Quality-of-Life Questionnaire-C30, version 3 (EORTC QLQ-C30). METHODS: Following completion of the questionnaires, patients were interviewed to assess the validity, clarity, relevance and comprehensiveness of the assessments. RESULTS: Overall, patients considered both the FACIT-Fatigue and EORTC QLQ-C30 instruments to be relevant and adequate in assessing the level of PNH-associated fatigue and other quality-of-life measures. The FACIT-Fatigue questionnaire was considered to be clear and to comprehensively cover PNH-related fatigue. The EORTC QLQ-C30 instrument was considered to be easy to understand, but of an overall lower relevance, although some differences between countries were observed. Patients suggested additional questions that could be incorporated into future EORTC QLQ-C30 versions to make it more relevant to PNH. CONCLUSIONS: This study confirms the validity of the FACIT-Fatigue and the EORTC QLQ-C30 questionnaires in this patient population and their routine use should be considered in the management of patients with PNH.

Is it time for a change? The case for early application of unrelated allo-SCT for severe aplastic anemia.

Severe aplastic anemia (SAA) is a BM failure syndrome in which allo-SCT remains a highly effective curative option. Its application remains limited by donor availability and by the potential for treatment-related morbidity and mortality. The improved outcomes with unrelated transplantation are a result of the advent of molecular donor-recipient matching, generation of effective novel conditioning regimens, improvement of supportive care and expansion of the donor registry. Decision making regarding the earlier use of unrelated transplant procedures is rapidly evolving. This paper reviews critical data relevant to these treatment options and recommends early consideration of related SCT for patients with SAA who show failure of immune suppressive therapy.

The role of the defective interfering particle DI9c in mucosal disease in cattle.

Mucosal disease occurs in cattle persistently infected with a noncytopathogenic strain of bovine viral diarrhoea virus (BVDVnc) following in utero infection. The disease can be initiated by superinfection with a cytopathogenic biotype (BVDVc) of the virus with antigenic "homology" to the persisting virus. A BVDVc isolated from a clinical case of mucosal disease has been discovered to consist of a defective interfering particle, DI9, and an associated BVDVnc helper virus. A defective virus corresponding to DI9 was recently recovered from an infectious cDNA clone and was named DI9c. To evaluate the role of DI9 in the pathogenesis of mucosal disease a two-part experimental study was carried out which included clinical, haematological, pathological and virological investigations. Eight of nine calves persistently infected with BVDVnc were experimentally inoculated with DI9c. The defective virus was propagated in cells preinfected with the same strain of virus used to persistently infect the calves in utero. The calves were euthanased on days 4, 7, 14, 21, 28, 40, 40 or 87 post inoculation. None of the inoculated animals developed classical mucosal disease, neither clinically nor pathologically. DI9c was not found in serum, nasal swab or tissue samples from the calves by observing cytopathogenic effect and/or using a polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. DI9c did not replicate to a detectable extent in these assays, and its participation in the pathogenesis of mucosal disease could not be proven.

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries.

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus.

Pestiviruses are positive-strand RNA viruses closely related to human hepatitis C virus. Gene expression of these viruses occurs via translation of a polyprotein, which is further processed by cellular and viral proteases. Here we report the formation of a stable complex between an as-yet-undescribed cellular J-domain protein, a member of the DnaJ-chaperone family, and pestiviral nonstructural protein NS2. Accordingly, we termed the cellular protein Jiv, for J-domain protein interacting with viral protein. Jiv has the potential to induce in trans one specific processing step in the viral polyprotein, namely, cleavage of NS2-3. Efficient generation of its cleavage product NS3 has previously been shown to be obligatory for the cytopathogenicity of the pestiviruses. Regulated expression of Jiv in cells infected with noncytopathogenic bovine viral diarrhea virus disclosed a direct correlation between the intracellular level of Jiv, the extent of NS2-3 cleavage, and pestiviral cytopathogenicity.

A family of closely related bovine viral diarrhea virus recombinants identified in an animal suffering from mucosal disease: new insights into the development of a lethal disease in cattle.

Induction of lethal mucosal disease (MD) in cattle is linked to the generation of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) in animals persistently infected with a noncytopathogenic BVDV. In most cases the cp variants are generated by recombination with cellular or viral sequences. BVDV was obtained from the serum of an MD animal and propagated in tissue culture without plaque purification. Analysis of cDNA clones established from RNA of these cells showed that apparently a variety of different viral RNAs were present. Seven of the cDNA clones contained a cellular sequence coding for light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This insertion had already been found in the cp virus JaCP obtained from the same animal and isolated by plaque purification. Analysis of further plaque-purified cp viruses showed that the diseased animal contained a family of closely related cp BVDV recombinants. A set of viruses with different duplications of viral sequences in their genomes and a variety of defective viral RNAs with deletions were found that all contained the LC3* insertion. For all the recombinants the 3' recombination sites and, in all but one case, also the 5' recombination sites between cellular and viral sequence were identical. Variation between the individual deduced genome structures resulted from different duplications or deletions of viral sequences located upstream of the cellular insertion. These results suggest that within the animal a primary recombinant with a genome containing the LC3* insertion was generated. In a trimming process a set of secondary virus recombinants was generated from this hypothetical primary recombined RNA. These secondary recombinants display genome structures that represent variations of the basic scheme already present in the primary recombinant. Apparently this trimming process that finally led to an outbreak of MD lasted a long time since recombined RNA with the basic genome structure of the cp viruses could be demonstrated in samples already taken a long time before outbreak of the disease.

The genetic basis for cytopathogenicity of pestiviruses.

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by their effects on tissue culture cells. Identification of cp bovine viral diarrhea virus (BVDV) has been frequently reported since antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system, only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed a second way for the development of a cp pestivirus which is based on the introduction of a set of point mutations within the NS2 gene.

Rabbit hemorrhagic disease virus: genome organization and polyprotein processing of a calicivirus studied after transient expression of cDNA constructs.

Rabbit hemorrhagic disease virus (RHDV) belongs to the family Caliciviridae. Studies on this virus are hampered by the lack of a convenient cell culture system. To study viral protein expression a cDNA construct containing the entire protein-coding region of the virus was established and used for transient expression studies. After metabolic labeling of transfected cells and immunoprecipitation with a set of RHDV-specific antisera a variety of polypeptides were identified and assigned to defined regions of the viral genome. The consensus sequences of already identified or putative proteolytic cleavage sites in the viral polyprotein were changed by the introduction of mutations into the expression construct. Expression of these mutated constructs and analysis of the protein patterns allowed us to identify novel cleavage sites in the polyprotein and revealed the first details regarding the order of polyprotein processing.

Better palpation of pain: reliability and validity of a new pressure pain protocol in TMD.

The purpose of this research was to employ a double-blind placebo control design of iontophoretically administered lidocaine to assess the test-retest reliability and validity of a magnitude matching procedure using a pressure algometer. In studies in the literature on assessment of pain in temporomandibular disorders (TMD), the pressure algometer has been found to be a reliable means of measuring pressure-pain threshold and a sensitive measure of treatment-response in the laboratory. However, previous research with this apparatus has not employed more sophisticated psychophysics. This study employs a magnitude matching psychophysical protocol to obtain a multidimensional pain report. Test-retest reliability over two occasions, 3-8 days apart, was found to be moderate for discriminability (r = .71, p < .01) but poor for response bias (r = .44). The validity study used iontophoresis as an anesthetic in a double-blind placebo and no-treatment control design. Although it was hypothesized that subjects in the anesthesia group would demonstrate reduced discriminability as compared with the control groups, no differences were found among the three groups on this measure. However, differences in response bias were found, with both the placebo control and no-treatment control groups differing from the experimental group but not from each other. Possible explanations and the implications of these findings are discussed.

Correlation between point mutations in NS2 and the viability and cytopathogenicity of Bovine viral diarrhea virus strain Oregon analyzed with an infectious cDNA clone.

Cytopathogenicity of Bovine viral diarrhea virus (BVDV) is correlated with expression of the nonstructural protein NS3, which can be generated by processing of a fusion protein termed NS2-3. For the cytopathogenic (cp) BVDV strain Oregon, NS2-3 processing is based on a set of point mutations within NS2. To analyze the correlation between NS2-3 cleavage and cytopathogenicity, a full-length cDNA clone composed of cDNA from BVDV Oregon and the utmost 5'- and 3'-terminal sequences of a published infectious BVDV clone was established. After transfection of RNA transcribed from this cDNA clone, infectious virus with similar growth characteristics to wild-type BVDV Oregon could be recovered that also exhibited a cytopathic effect. Based on this cDNA construct and published cp and noncp infectious clones, chimeric full-length cDNA clones were constructed. Analysis of the recovered viruses demonstrated that the presence of the NS2 gene of BVDV Oregon in a chimeric construct is sufficient for NS2-3 processing and a cp phenotype. Since previous studies had revealed that the amino acid serine at position 1555 of BVDV Oregon plays an important role in efficient NS2-3 cleavage, mutants of BVDV Oregon with different amino acids at this position were constructed. Some of these mutants showed NS2-3 cleavage efficiencies in the range of the wild-type sequence and allowed the recovery of viruses that behaved similarly to wild-type virus with regard to growth characteristics and cytopathogenicity. In contrast, other mutants with considerably reduced NS2-3 cleavage efficiencies propagated much more slowly and reverted to viruses expressing polyproteins with sequences allowing efficient NS2-3 cleavage. These viruses apparently induced cytopathic effects only after reversion.

Mutations abrogating the RNase activity in glycoprotein E(rns) of the pestivirus classical swine fever virus lead to virus attenuation.

Classical swine fever (CSF) is a severe hemorrhagic disease of swine caused by the pestivirus CSF virus (CSFV). Amino acid exchanges or deletions introduced by site-directed mutagenesis into the putative active site of the RNase residing in the glycoprotein E(rns) of CSFV abolished the enzymatic activity of this protein, as demonstrated with an RNase test suitable for detection of the enzymatic activity in crude cell extracts. Incorporation of the altered sequences into an infectious CSFV clone resulted in recovery of viable viruses upon RNA transfection, except for a variant displaying a deletion of the histidine codon at position 297 of the long open reading frame. These RNase-negative virus mutants displayed growth characteristics in tissue culture that were undistinguishable from wild-type virus and were stable for at least seven passages. In contrast to animals inoculated with an RNase-positive control virus, infection of piglets with an RNase-negative mutant containing a deletion of the histidine codon 346 of the open reading frame did not lead to CSF. Neither fever nor extended viremia could be detected. Animals infected with this mutant did not show decrease of peripheral B cells, a characteristic feature of CSF in swine. Animal experiments with four other mutants with either exchanges of codons 297 or 346 or double exchanges of both codons 297 and 346 showed that all these RNase-negative mutants were attenuated. All viruses with mutations affecting codon 346 were completely apathogenic, whereas those containing only changes of codon 297 consistently induced clinical symptoms for several days, followed by sudden recovery. Analyses of reisolated viruses gave no indication for the presence of revertants in the infected animals.

High prevalence of GB virus C in Brazil and molecular evidence for intrafamilial transmission.

The prevalence of GB virus C (GBV-C) in candidate Brazilian blood donors with normal and elevated alanine aminotransferase levels was found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected with HCV. Molecular characterization of GBV-C by partial sequencing of the NS3 region showed clustering between members of a single family, implying intrafamilial transmission. In conclusion, these results together suggest that contagion mechanisms which facilitate intrafamilial transmission of GBV-C may partially explain the high prevalence of viremic carriers worldwide.

Pathogenesis of mucosal disease, a deadly disease of cattle caused by a pestivirus.

BACKGROUND: Two biotypes of pestiviruses, cytopathogenic (cp) and non-cytopathogenic (noncp) viruses, are distinguished by their effects on tissue culture cells. In contrast to the bovine viral diarrhoea virus (BVDV) system, only a few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been described. Antigenically closely related noncp and cp BVDV can be isolated from cattle with fatal mucosal disease (MD) and are called a virus pair. The generation of cp BVDV in an animal persistently infected with noncp BVDV is regarded as causative for the development of MD. OBJECTIVES: To analyse viral pairs of BVDV at the molecular level and thereby identify differences between the viruses of each pair. STUDY DESIGN: BVDV pairs were isolated from several animals coming down with MD; the genomes of the respective BVD viruses were sequenced on cDNA level. Studies concerning the polyprotein processing of each strain were carried out. RESULTS: Molecular analysis of BVDV pairs demonstrated a linkage between RNA recombination, generation of NS3 and the onset of fatal MD. CONCLUSION: The molecular analysis of BVDV pairs revealed that the respective cp strains arise by RNA recombination from noncp viruses.

Isolation and characterization of cytopathogenic classical swine fever virus (CSFV).

Two new classical swine fever virus (CSFV) isolates obtained from naturally infected swine were found to exhibit a cytopathogenic (cp) phenotype. According to their reactivity with monoclonal antibodies (mabs) the isolates cpBW1 and cpMVP1 were classified as antigenic types "Lothringen'92" and "Flandern'90", respectively. In Northern blot analyses and PCR assays CSFV RNA of subgenomic length was detected in infected cells indicating the presence of defective interfering particles. Nucleotide sequencing of cDNA fragments obtained by reverse transcription (RT)-PCR allowed to localize in the genomes of the cpMVP1 and cpBW1 DIs deletions of 4,764 and 4,746, respectively. From the latter isolate a noncytopathogenic (noncp) biotype was obtained by cloning in cell culture. CSFV cpBW1 and a noncp isolate were used separately for infection of pigs. Viremia occurred much quicker after infection with cp virus and clinical signs were more prominent compared to animals infected with noncp virus.

Structure of the human biotinidase gene.

Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin. We have determined the structure of the human biotinidase gene. A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization. To obtain a clone containing the most 5' exon of the biotinidase cDNA, a human PAC library by PCR was screened. The human biotinidase gene is organized into four exons and spans at least 23 kb. The 5'-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element. The region from nt -600 to +400 has features of a CpG island and resembles a housekeeping gene promoter. The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency.

Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus.

The calicivirus rabbit hemorrhagic disease virus (RHDV), which replicates predominantly in the livers of infected rabbits, cannot be propagated in tissue culture. To enable the performance of in vitro studies, rabbit hepatocytes were isolated by liver perfusion and gradient centrifugation. After inoculation with purified RHDV, more than 50% of the cells proved to be infected. Protein analyses led to the detection of 13 RHDV-specific polypeptides within the infected cells. These proteins were assigned to defined regions of the viral genome, resulting in a refined model of RHDV genome organization.