Functional and structural assessment of patients with and without persistent pain after thoracotomy.

BACKGROUND: Persistent pain is frequent after thoracotomy, with a reported prevalence of up to 60%. It remains unclear why some patients develop pain, whereas others do not. We therefore examined patients with and without pain after thoracotomy to identify pathophysiological contributors to persistent pain. METHODS: Twenty patients with persistent pain, 12 patients without pain and 20 healthy controls underwent detailed functional and structural assessment including psychometric and neuropathic pain questionnaires, bedside examination for pinprick hyperalgesia and brush allodynia, quantitative sensory testing according to the protocol of the German Research Network on Neuropathic Pain, measurement of capsaicin-evoked flare response, intradermal nerve density as determined by skin biopsies and laser- and heat-evoked potentials. RESULTS: Bedside testing revealed evoked pain in 16 of 20 patients with pain, but only in 2 of 12 patients without pain (p < 0.001). Quantitative sensory testing showed increased mechanical pain sensitivity (p = 0.018) on the operated side in patients with pain, but there were no differences between the two patient groups with regard to intradermal nerve fibre density, area and flux following capsaicin application and laser- and heat-evoked potentials. CONCLUSION: Different and individual pathophysiological mechanisms of pain may obscure the clinical picture and thus preclude identification of a specific pain profile in patients with persistent post-thoracotomy pain. SIGNIFICANCE: Evoked pain is more frequent in patients with pain. Assessment of intradermal nerve density, capsaicin-induced flare response and contact and laser heat-evoked potentials revealed no differences between pain patients and pain-free patients.

Assessment of acute oxaliplatin-induced cold allodynia: a pilot study.

BACKGROUND: Following oxaliplatin treatment, acute neurotoxicity symptoms are suggested to be correlated with both the development and degree of chronic neuropathy. AIMS: The aim of this clinical commentary was to examine different methods to assess acute cold allodynia and dysesthesia in patients treated with adjuvant oxaliplatin. METHODS: Nine patients over the age of 18 years scheduled for standard adjuvant treatment with capecitabine and oxaliplatin were included. Patients were asked to come for two visits: a baseline visit before and a follow-up visit within 5 days after treatment. Patients were examined with questionnaires, thermal tests, and the thermal grill. RESULTS: All patients reported neurotoxicity, and they all had abnormal cold sensitivity. The only significant changes observed were increased ratings of pain, unpleasantness, and pricking sensations to holding a ~8°C metal cylinder for 10 s and an increased intensity of unpleasantness and pricking sensation to the 20-s contact with the 10°C plates of the thermal grill on the palmar hand. CONCLUSIONS: he results showed that the palm of the hand is the most sensitive part of the body when detecting oxaliplatin-induced cold allodynia, and the use of a cold metal cylinder seems as a promising sensitive method.

The effect of nerve compression and capsaicin on contact heat-evoked potentials related to Aδ- and C-fibers.

Brief noxious heat stimuli activate Aδ- and C-fibers and allow contact heat-evoked potentials (CHEPs) to be recorded from the scalp. Under normal conditions, only late responses related to Aδ-fibers can be recorded. This study aimed to demonstrate C-fiber responses to contact heat stimuli. A preferential A-fiber compression blockade of the superficial radial nerve was applied in 22 healthy subjects. Quality and intensity of heat-evoked pain and CHEPs were examined at baseline, during nerve compression, and during nerve compression with simultaneous application of topical capsaicin (5%). During the A-fiber block, three subjects had CHEPs with latencies below 400 ms, eight subjects within 400-800 ms and six subjects (29%) later than 800 ms. Pain intensity to contact heat stimuli after compression was reduced and fewer subjects reported the heat stimuli as stinging. Following nerve compression and capsaicin application, ultralate CHEPs with latencies >800 ms could be recorded in 13 subjects (62%), pain intensity to the contact heat stimuli was increased and the warm/hot-burning pain quality became more intense. The main results of our study are the demonstration of ultralate C-fiber-related CHEPs following A-fiber blockade in 29% of healthy subjects increasing to 62% when the blockade was combined with capsaicin. After blockade of Aδ-fibers we recorded responses with latencies in the range between the latencies of Aδ- and C-fibers suggesting release of Aδ-fibers with slower conduction velocity than normally recorded with CHEPs.

Increased contact heat pain and shortened latencies of contact heat evoked potentials following capsaicin-induced heat hyperalgesia.

OBJECTIVE: To examine changes in contact heat evoked potentials (CHEPs) and perceived pain intensity following acute sensitization with topical capsaicin. METHODS: CHEPs were recorded before and after 20 min of topical capsaicin application (200 µl, 5%) during skin warming in 22 healthy subjects. To evaluate the sequence effects and skin warming on CHEPs, 10 of these subjects also participated in a control study. RESULTS: Topical capsaicin yielded an increase in contact heat evoked pain ratings (p < 0.0001) and a shortening in N2 latency from a mean 345.2 ± 37.2 ms to 310.2 ± 38.5 ms recorded from the vertex position (p = 0.003, paired t-test). No difference was found in the N2-P2 peak-to-peak amplitude (p = 0.83). These results were unchanged after controlling for sequence effects and skin warming. Following capsaicin, ultralate CHEPs (N2a latencies 970-1352 ms) were recorded in three subjects. CONCLUSIONS: Our study showed a decrease in late CHEPs latencies and appearance of ultralate potentials compatible with sensitization of Aδ fibers and C fibers. SIGNIFICANCE: Contact heat may be a useful tool to assess sensitization of the pain system.

MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment.

Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+ CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.

Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury.

While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.

Mice with spontaneous pancreatic cancer naturally develop MUC-1-specific CTLs that eradicate tumors when adoptively transferred.

Pancreatic cancer is a highly aggressive, treatment refractory cancer and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas overexpress altered forms of a tumor-specific Ag, mucin 1 (MUC1; an epithelial mucin glycoprotein), which is a potential target for immunotherapy. We have established a clinically relevant animal model for pancreatic cancer by developing a double transgenic mouse model (called MET) that expresses human MUC1 as self molecule and develops spontaneous tumors of the pancreas. These mice exhibit acinar cell dysplasia at birth, which progresses to microadenomas and acinar cell carcinomas. The tumors express large amounts of underglycosylated MUC1 similar to humans. Tumor-bearing MET mice develop low affinity MUC1-specific CTLs that have no effect on the spontaneously occurring pancreatic tumors in vivo. However, adoptive transfer of these CTLs was able to completely eradicate MUC1-expressing injectable tumors in MUC1 transgenic mice, and these mice developed long-term immunity. These CTLs were MHC class I restricted and recognized peptide epitopes in the immunodominant tandem repeat region of MUC1. The MET mice appropriately mimic the human condition and are an excellent model with which to elucidate the native immune responses that develop during tumor progression and to develop effective antitumor vaccine strategies.

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein.

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF-TBP binding depended on the C-terminal 'acidic tail' of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

Substitution of the degenerate smooth muscle (SM) alpha-actin CC(A/T-rich)6GG elements with c-fos serum response elements results in increased basal expression but relaxed SM cell specificity and reduced angiotensin II inducibility.

We have previously demonstrated that both CC(A/T-rich)6GG (CArG) elements A and B of the smooth muscle (SM) alpha-actin promoter are required for smooth muscle cell (SMC)-specific expression and angiotensin II (AII)-induced stimulation. Moreover, results provided evidence that AII responsiveness of SM alpha-actin was at least partially dependent on modulation of serum response factor (SRF) binding to the SM alpha-actin CArGs by the homeodomain containing protein, MHox. The goal of the present study was to investigate whether the degeneracy of the SM alpha-actin CArGs (both contain a Gua or Cyt substitution in their A/T-rich center) and their reduced SRF binding activity as compared with c-fos serum response element (SRE) is important for conferring cell type-specific expression and AII responsiveness. Transient transfection assays using SM alpha-actin reporter gene constructs in which the endogenous SM alpha-actin CArGs were replaced by c-fos SREs demonstrated the following: 1) relaxation of cell-specific expression, 2) a 50% reduction in AII responsiveness, and 3) reduced ability to be transactivated by MHox. In addition, we also showed that the position of the SM alpha-actin CArGs was important in that interchanging them abolished both basal and AII-induced activities. Taken together, these results suggest that the reduced SRF binding activities of the SM alpha-actin CArGs and CArG positional context contribute to SMC-specific expression of SM alpha-actin as well as maximal AII responsiveness.

Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells.

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.

Mucin mRNA expression in normal and vasomotor inferior turbinates.

Mucins are the major glycoprotein component of respiratory tract secretions. Little is known about their expression in the upper respiratory tract. In order to define this expression, in situ hybridization was performed on 19 normal and 4 vasomotor rhinitis (VMR) inferior turbinates to identify mucin mRNA. MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 were expressed in both the normal and VMR turbinates. MUC4 and MUC5AC were the most highly expressed mucins. MUC1, MUC2, MUC4, and MUC5AC were expressed mainly by the epithelial border, whereas MUC5B and MUC7 were expressed by the submucosal glands. MUC1 and MUC4 exhibited a diffuse expression by multiple cell types along the mucosal border, whereas MUC2 and MUC5AC expression appeared to be limited to a subpopulation of epithelial cells, most likely goblet cells. Although MUC1, MUC4, and MUC5AC showed sporadic submucosal glandular expression, MUC5B and MUC7 appeared to be the predominant submucosal gland mucins in the inferior turbinates. MUC3 and MUC6 expression, which have been found primarily in the gastric mucosa, were not seen in any of the inferior turbinate samples examined. The only difference seen between normal and VMR turbinates was a slight decrease in MUC1 expression in the VMR group. The variety of mucins expressed and the diversity of their expression patterns may have significance in terms of the rheologic and particle clearance properties of nasal secretions. Understanding the expression patterns in normal turbinates will serve as the foundation for further study of these mucins in disease states.

A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements.

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGFbeta) increases smooth muscle (SM) alpha-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha-actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.

Expression of the smooth muscle myosin heavy chain gene is regulated by a negative-acting GC-rich element located between two positive-acting serum response factor-binding elements.

To identify cis- and trans-acting factors that regulate smooth muscle-specific gene expression, we studied the smooth muscle myosin heavy chain gene, a rigorous marker of differentiated smooth muscle. A comparison of smooth muscle myosin heavy chain promoter sequences from multiple species revealed the presence of a highly conserved 227-base pair domain (nucleotides -1321 to -1095 in rat). Results of a deletion analysis of a 4.3-kilobase pair segment of the rat promoter (nucleotides -4220 to +88) demonstrated that this domain was necessary for maximal transcriptional activity in smooth muscle cells. Gel-shift analysis and site-directed mutagenesis demonstrated that one true CArG and another CArG-like element contained within this domain were both recognized by the serum response factor and were both required for the positive activity attributable to this domain. Additional studies demonstrated that mutation of a GC-rich sequence within the 227-base pair conserved domain resulted in a nearly 100% increase in transcriptional activity. Gel-shift analysis showed that this GC-rich repressor element was recognized by both Sp1 and Sp3. These data demonstrate that transcriptional control of the smooth muscle myosin heavy chain gene is highly complex, involving both negative and positive regulatory elements, including CArG sequences found in the promoters of multiple smooth muscle differentiation marker genes.

Molecular regulation of smooth muscle cell differentiation.

REGULATION OF SMOOTH MUSCLE CELL (SMC) GROWTH: Accelerated growth of SMC is known to play an integral role in atherosclerotic lesion formation as well as post-angioplasty restenosis, and is a characteristic feature in arteries of hypertensive patients and animals. There has thus been extensive interest in defining both positive and negative regulators of SMC growth, and many factors have been identified that may play a role in this process. REGULATION OF DIFFERENTIATED SMC: Despite clear evidence that the differentiated state of the intimal SMC is altered in atherosclerotic disease, and that this is likely to play a key role in lesion development, relatively little is known about the mechanisms and factors that regulate the differentiated state of SMC. Extrinsic factors (local environmental cues) make an important contribution to the regulation of the differentiated state of the vascular SMC. Molecular mechanisms control the expression of genes encoding for proteins such as smooth muscle alpha-actin and smooth muscle myosin heavy chain that are selective or specific for SMC, and which are required for the SMC principal differentiated function, contraction.

In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication.

Using in organello footprint analysis, we demonstrate that within human placental mitochondria there is a high level of protein-DNA binding at regularly phased intervals throughout a 500-bp region encompassing the D-loop DNA origins and two promoter regions. Comparison with in vitro DNase I protection studies indicates that this protein-DNA interaction is due to non-sequence-specific binding by human mitochondrial transcription factor A (h-mtTFA). Since h-mtTFA can bend and wrap DNA, like its yeast counterpart ABF2, a primary function of h-mtTFA appears to be specific packaging of the mitochondrial DNA control region in vivo. Intervals of protein binding coincide with the spacing of the RNA start sites and prominent D-loop DNA 5' ends, suggesting a role for phased h-mtTFA binding in defining transcription and H-strand DNA replication origins. Significant protein-DNA interaction was also observed within the human homolog of conserved sequence block 1, both in organello and in vitro, using purified h-mtTFA.

Sequence conservation of an avian centromeric repeated DNA component.

The approximately 190-bp centromeric repeat monomers of the spur-winged lapwing (Vanellus spinosus, Charadriidae), the Chilean flamingo (Phoenicopterus chilensis, Phoenicopteridae), the sarus crane (Grus antigone, Gruidae), parrots (Psittacidae), waterfowl (Anatidae), and the merlin (Falco columbarius, Falconidae) contain elements that are interspecifically highly variable, as well as elements (trinucleotides and higher order oligonucleotides) that are highly conserved in sequence and relative location within the repeat. Such conservation suggests that the centromeric repeats of these avian species have evolved from a common ancestral sequence that may date from very early stages of avian radiation.

In vivo and in vitro evidence for slipped mispairing in mammalian mitochondria.

Slipped mispairing between repeated sequences during DNA replication is an important mutagenic event. It is one of several suggested mechanisms thought to be responsible for generating polymorphic regions and large-scale deletions found in mammalian mitochondrial DNA. In the porcine mitochondrial genome, a domain carrying a 10-bp tandemly repeated sequence displays a unique in vivo pattern of repeat copy number polymorphs. Upon passage in Escherichia coli, a recombinant plasmid containing this domain also displays a unique polymorphic pattern that is different from that seen in the animal. To test the hypothesis that these polymorphisms were slippage induced and that the different polymorphic patterns reflected differences in modes of replication, we performed a series of in vitro primer extension reactions. By utilizing either single- or double-stranded templates containing the repeat domain we were able to correlate in vitro generated repeat polymorphism patterns with those seen in the mitochondria or the bacteria, respectively, thus providing experimental evidence that slippage replication is responsible for a major class of mammalian mutations.