Sensitivity, specificity, and accuracy of molecular profiling on circulating cell-free DNA in refractory or relapsed multiple myeloma patients, results of MM-EP1 study.

As a promising alternative to bone marrow aspiration (BMA), mutational profiling on blood-derived circulating cell-free tumor DNA (cfDNA) is a harmless and simple technique to monitor molecular response and treatment resistance of patients with refractory/relapsed multiple myeloma (R/R MM). We evaluated the sensitivity and specificity of cfDNA compared to BMA CD138 positive myeloma plasma cells (PCs) in a series of 45 R/R MM patients using the 29-gene targeted panel (AmpliSeq) NGS. KRAS, NRAS, FAM46C, DIS3, and TP53 were the most frequently mutated genes. The average sensitivity and specificity of cfDNA detection were 65% and 97%, respectively. The concordance per gene between the two samples was good to excellent according to Cohen's κ coefficients interpretation. An increased number of mutations detected in cfDNA were associated with a decreased overall survival. In conclusion, we demonstrated cfDNA NGS analysis feasibility and accuracy in R/R MM patients who may benefit from early phase clinical trial.

Acute intermittent hypoxia enhances regeneration of surgically repaired peripheral nerves in a manner akin to electrical stimulation.

The intrinsic repair response of injured peripheral neurons is enhanced by brief electrical stimulation (ES) at time of surgical repair, resulting in improved regeneration in rodents and humans. However, ES is invasive. Acute intermittent hypoxia (AIH) - breathing alternate cycles of regular air and air with ~50% normal oxygen levels (11% O2), considered mild hypoxia, is an emerging, promising non-invasive therapy that promotes motor function in spinal cord injured rats and humans. AIH can increase neural activity and under moderately severe hypoxic conditions improves repair of peripherally crushed nerves in mice. Thus, we posited an AIH paradigm similar to that used clinically for spinal cord injury, will improve surgically repaired peripheral nerves akin to ES, including an impact on regeneration-associated gene (RAG) expression-a predictor of growth states. Alterations in early RAG expression were examined in adult male Lewis rats that underwent tibial nerve coaptation repair with either 2 days AIH or normoxia control treatment begun on day 2 post-repair, or 1 h ES treatment (20 Hz) at time of repair. Three days post-repair, AIH or ES treatments effected significant and parallel elevated RAG expression relative to normoxia control at the level of injured sensory and motor neuron cell bodies and proximal axon front. These parallel impacts on RAG expression were coupled with significant improvements in later indices of regeneration, namely enhanced myelination and increased numbers of newly myelinated fibers detected 20 mm distal to the tibial nerve repair site or sensory and motor neurons retrogradely labeled 28 mm distal to the repair site, both at 25 days post nerve repair; and improved return of toe spread function 5-10 weeks post-repair. Collectively, AIH mirrors many beneficial effects of ES on peripheral nerve repair outcomes. This highlights its potential for clinical translation as a non-invasive means to effect improved regeneration of injured peripheral nerves.

Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve.

The neurotrophin family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Previous studies have demonstrated that expression of NGF and its low-affinity receptor is induced in nonneuronal cells of the distal segment of the transected sciatic nerve suggesting a role for NGF during axonal regeneration (Johnson, E. M., M. Taniuchi, and P. S. DeStefano. 1988. Trends Neurosci. 11:299-304). To assess the role of the other neurotrophins and the members of the family of Trk signaling neurotrophin receptors, we have here quantified the levels of mRNAs for BDNF, NT-3, and NT-4 as well as mRNAs for trkA, trkB, and trkC at different times after transection of the sciatic nerve in adult rats. A marked increase of BDNF and NT-4 mRNAs in the distal segment of the sciatic nerve was seen 2 wk after the lesion. The increase in BDNF mRNA was mediated by a selective activation of the BDNF exon IV promoter and adrenalectomy attenuated this increase by 50%. NT-3 mRNA, on the other hand, decreased shortly after the transection but returned to control levels 2 wk later. In Schwann cells ensheathing the sciatic nerve, only trkB mRNA encoding truncated TrkB receptors was detected with reduced levels in the distal part of the lesioned nerve. Similar results were seen using a probe that detects all forms of trkC mRNA. In the denervated gastrocnemius muscle, the level of BDNF mRNA increased, NT-3 mRNA did not change, while NT-4 mRNA decreased. In the spinal cord, only small changes were seen in the levels of neutrophin and trk mRNAs. These results show that expression of mRNAs for neurotrophins and their Trk receptors is differentially regulated after a peripheral nerve injury. Based on these results a model is presented for how the different neurotrophins could cooperate to promote regeneration of injured peripheral nerves.

Reaching training in rats with spinal cord injury promotes plasticity and task specific recovery.

In the current study we examined the effects of training in adult rats with a cervical spinal cord injury (SCI). One group of rats received 6 weeks of training in a single pellet reaching task immediately after injury, while a second group did not receive training. Following this period changes in cortical levels of BDNF and GAP-43 were analysed in trained and untrained animals and in a group with training but no injury. In another group of rats, functional recovery was analysed in the reaching task and when walking on a horizontal ladder. Thereupon, the cortical forelimb area was electrophysiologically examined using micro-stimulation followed by tracing of the lesioned corticospinal tract (CST). We found that trained rats improved substantially in the reaching task, when compared to their untrained counterparts. Trained rats however, performed significantly worse with their injured forelimb when walking on a horizontal ladder. In parallel to the improved recovery in the trained task, we found that the cortical area where wrist movements could be evoked by micro-stimulation expanded in trained rats in comparison to both untrained and uninjured rats. Furthermore, collateral sprouting of lesioned CST fibres rostral to the injury was increased in trained rats. Post-injury training was also found to increase cortical levels of GAP-43 but not BDNF. In conclusion we show that training of a reaching task promotes recovery of the trained task following partial SCI by enhancing plasticity at various levels of the central nervous system (CNS), but may come at the cost of an untrained task.

Electrical stimulation as a conditioning strategy for promoting and accelerating peripheral nerve regeneration.

The delivery of a nerve insult (a "conditioning lesion") prior to a subsequent test lesion increases the number of regenerating axons and accelerates the speed of regeneration from the test site. A major barrier to clinical translation is the lack of an ethically acceptable and clinically feasible method of conditioning that does not further damage the nerve. Conditioning electrical stimulation (CES), a non-injurious intervention, has previously been shown to improve neurite outgrowth in vitro. In this study, we examined whether CES upregulates regeneration-associated gene (RAG) expression and promotes nerve regeneration in vivo, similar to a traditional nerve crush conditioning lesion (CCL). Adult rats were divided into four cohorts based on conditioning treatment to the common peroneal (fibular) nerve: i) CES (1h, 20Hz); ii) CCL (10s crush); iii) sham CES (1h, 0Hz); or iv) naïve (unconditioned). Immunofluorescence and qRT-PCR revealed significant RAG upregulation in the dorsal root ganglia of both CES and CCL animals, evident at 3-14days post-conditioning. To mimic a clinical microsurgical nerve repair, all cohorts underwent a common peroneal nerve cut and coaptation one week following conditioning. Both CES and CCL animals increased the length of nerve regeneration (3.8-fold) as well as the total number of regenerating axons (2.2-fold), compared to the sham and naïve-conditioned animals (p<0.001). These data support CES as a non-injurious conditioning paradigm that is comparable to a traditional CCL and is therefore a novel means to potentially enhance peripheral nerve repair in the clinical setting.

Neurotrophin-3 attenuates galanin expression in the chronic constriction injury model of neuropathic pain.

We have recently shown that exogenous neurotrophin-3 (NT-3) acts antagonistically to nerve growth factor (NGF) in regulation of nociceptor phenotype in intact neurons and suppresses thermal hyperalgesia and expression of molecules complicit in this behavioral response induced by chronic constriction injury (CCI) of the sciatic nerve. The present study examines whether there is a global influence of NT-3 in mitigating alterations in peptide and NGF receptor expression; molecules believed to also contribute to CCI-associated pain. Thus, the influence of NT-3 on phenotypic changes in dorsal root ganglion (DRG) neurons in rats coincident with CCI was examined using in situ hybridization. Seven days following injury, the incidence of expression of the neuropeptides galanin and pituitary adenylate cyclase-activating polypeptide (PACAP) was increased in L5 sensory neurons ipsilateral to the injury from 12% to 60% and 16% to 37% respectively, in addition to an increased level of expression. In contrast, there was no consistent significant change in tropomyosin-related kinase A (trkA) expression following CCI. Intrathecal infusion of NT-3 globally mitigated both the increased incidence and elevated levels of galanin messenger RNA (mRNA) expression observed following CCI, reducing the former from 60% to 39%. NT-3 infusion resulted in a limited reduction in the incidence and level of neuronal PACAP in medium to large size, but not small size, DRG neurons. NT-3 had no significant net effect on CCI-induced alterations in trkA mRNA expression.

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures.

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.

Nitric oxide synthase activity and expression in experimental diabetic neuropathy.

The changes of nitric oxide synthase (NOS) activity and expression in experimental diabetic neuropathy have not been examined. Increases in ganglia NOS might be similar to those that follow axotomy, whereas declines in endothelial NOS (eNOS) and immunological NOS (iNOS) might explain dysfunction of microvessels or macrophages. In this work, we studied NOS activity in lumbar dorsal root ganglia (DRG) of rats with both short- and long-term experimental streptozotocin-induced diabetes and correlated it with expression of each of the 3 NOS isoforms. NOS enzymatic activity in DRG increased after 12 months of diabetes. This increase, however, was not accompanied by an increase in neuronal NOS immunohistochemistry or mRNA. Immunohistochemical and RT-PCR studies did not identify changes of eNOS expression in 12-month sciatic nerves or DRG from diabetics. Two-month diabetic DRG had increased eNOS mRNA and there was novel eNOS labeling of capsular DRG and perineurial cells. iNOS mRNA levels were lower in diabetics at both time points in peripheral nerves but were unchanged in DRG. Diabetic ganglia showed an increase in NOS activity not explained by novel NOS isoform synthesis. The increases may compensate for NO "quenching" by endproducts of glycosylation. Declines in iNOS may indicate impaired macrophage function.

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy.

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences.

Anatomical evidence supporting the potential for modulation by multiple neurotrophins in the majority of adult lumbar sensory neurons.

Neurotrophins exert effects on sensory neurons through receptor tyrosine kinases (trks) and a common neurotrophin receptor (p75). Quantitative in situ hybridization studies were performed on serial sections to identify neurons expressing single or multiple neurotrophin trk receptor mRNA(s) in adult lumbar dorsal root ganglion (DRG) in order to examine the possibility of multi-neurotrophin modulation of phenotype via different trk receptors or various trk isoforms. Expression of mRNA encoding trkA, trkB, trkC, or p75 is restricted to select subpopulations representing approximately 41%, 33%, 43%, and 79% of DRG neurons, respectively. Colocalization studies reveal that approximately 10% of DRG neurons coexpress trkA and trkB mRNA; 19% coexpress trkA and trkC mRNA; and 18% coexpress trkB and trkC mRNA. Trilocalization of all three trk mRNAs is rare, with approximately 3-4% of neurons in this category. Overall incidence of expression of more than one full length trk mRNA occurs in approximately 40% of DRG neurons, whereas expression of individual trk mRNA is found in approximately 34%. Full length trk receptor mRNA is rarely detected without p75, implicating the latter in neuronal response to neurotrophins. Examination of two full-length isoforms of trkA reveal that they are coexpressed with relative levels of expression positively correlated. TrkC mRNAs corresponding to 14- or 39-amino acid insert isoforms colocalize with the non-insert trkC isoform, but the converse is not necessarily true. The data suggest that substantial subpopulations of adult sensory neurons may be modulated through interactions with multiple neurotrophins, the consequences of which are largely unknown.

Plasticity of NO synthase expression in the nervous and endocrine systems.

Using immunohistochemistry and in situ hybridization the effect of nerve injury and of hormones was analysed in sensory and hypothalamic systems and in the pituitary gland. After peripheral axotomy a marked increase in NOS protein and mRNA levels was observed in dorsal root ganglia, the trigeminal ganglion and a less dramatic effect in the nodose ganglia. This effect lasted in the dorsal root ganglion neurons for at least 10 weeks. In the hypothalamic magnocellular neurons a transient increase was observed in the paraventricular and supraoptic nuclei. A similar effect was also seen after salt loading. In the anterior pituitary gland NOS was expressed in gonadotrophs and folliculo-stellate cells. Castration markedly increased NOS levels in the anterior lobe, and this could be counteracted by steroid hormone replacement. Thus, the present results show that the constitutive, neuronal NOS can be dramatically regulated in response to various manipulations, suggesting an important involvement of NO in these situations.

Nerve growth factor induces functional nicotinic acetylcholine receptors on rat sensory neurons in culture.

Neonatal sensory neurons from rat nodose ganglia express nicotinic acetylcholine receptors when grown in tissue culture without other cell types. The present study investigates the role of nerve growth factor in inducing these receptors. Nerve growth factor has little effect on the growth and survival of nodose neurons in culture, although most neurons were found by quantitative radioautography to have high-affinity nerve growth factor receptors. Nerve growth factor strongly influenced the expression of nicotinic receptors on these neurons: the proportion of acetylcholine-sensitive neurons was approximately 60% in cultures with nerve growth factor compared with 15% in cultures grown without nerve growth factor. The proportion of acetylcholine-sensitive neurons increased over the first week, plateaued by day 12 and remained high for at least three weeks. In contrast, without NGF, the proportion of acetylcholine-sensitive neurons was low throughout the three-week period. The results indicate that nerve growth factor is an important factor in promoting nicotinic receptors on these neurons in culture.

Effect of anti-nerve growth factor treatment on pituitary adenylate cyclase activating polypeptide expression in adult sensory neurons exposed to adjuvant induced inflammation.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) is increased in sensory neurons exposed to adjuvant induced peripheral inflammation. Local elevation in expression of the neurotrophin nerve growth factor (NGF) is a main factor contributing to the neuronal response to inflammation. This study examines the role of endogenous NGF in inflammation-associated increases in PACAP expression using the adjuvant-induced peripheral inflammation model with or without systemic administration of antibodies against NGF. Quantitative in situ hybridization was used to detect changes in neuronal PACAP mRNA expression and to correlate this expression with neuronal mRNA expression of the NGF receptor tyrosine kinase (trk) A. The results from this study show that inflammation triggered increases in PACAP expression occurs in small- to medium-sized dorsal root ganglion (DRG) neurons that also express trkA, and that this elevation in PACAP expression is prevented by systemic injection of anti-NGF. This supports a role for NGF as a positive regulator of PACAP expression during inflammation.

Localization of neurotrophin receptors in olfactory epithelium and bulb.

We used in situ hybridization to localize trk, trkB and trkC mRNA, in rat and cat olfactory bulb. Expression of mRNA encoding truncated trkB receptors was seen in all layers, while only very modest full-length trkB expression could be detected. trkC hybridization was seen in all layers, most dense in the mitral cell layer. The localization of full-length tyrosine kinase trkB receptor in olfactory bulb and epithelium was examined with immunohistochemistry. trkB-like immunoreactivity was seen in the fila olfactoria, epithelium and in vitro, in olfactory sensory neurones. Since BDNF is expressed by olfactory sensory neurone target cells in the olfactory bulb, these data suggest that BDNF may act as a target derived neurotrophic factor in the primary olfactory system.

trkC expression in the injured rat spinal cord.

Reactive non-neuronal cells express high levels of low-affinity neurotrophin receptor and truncated trkB receptors after spinal cord injury. Here we report that descending nerve fibres in the rat lateral spinal cord column show strong trkC-like immunoreactivity after traumatic spinal cord lesions in the adult rat. No change in trkC expression by glial cells could be detected by immunohistochemistry or in situ hybridization at the lesion site. The data suggest that regeneration of descending spinal cord axons could be encouraged by the trkC ligand, neurotrophin 3.

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection.

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.

Properties of receptors for nerve growth factor in the mature rat nervous system.

The binding properties of receptors for nerve growth factor (NGF) in the adult rat nervous system have been analyzed in membrane fractions from discrete regions and in radioautographs of tissue sections. Steady-state experiments with both preparations revealed specific binding of radioiodinated NGF that was heterogeneous in distribution and affinity. Of the regions in the central nervous system that were sampled, the dorsal spinal cord and basal forebrain were richest in NGF receptor. By 4 independent methods of analysis a high-affinity binding site was detected in the basal forebrain with half-maximal saturation estimated at 20-60 pM NGF. Binding at lower affinity was also seen but difficult to analyze quantitatively. Cross-linking studies followed by electrophoresis showed the NGF receptor in the rat basal forebrain to have an apparent molecular weight of 90 kDa. The binding and molecular properties of NGF receptors on adult mammalian neurons resemble those described on other NGF-responsive cells.

Up-regulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain in the rat.

We examined the distribution of mRNA for the peptide cholecystokinin (CCK) with in situ hybridization in adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve, as well as the effect of systemic CI 988, a selective antagonist of the CCK type B receptor, applied alone or in combination with intrathecal (i.t.) morphine, on the self-mutilating behavior of rats (autotomy) after axotomy, a sign of neuropathic pain and/or dysesthesia. There was a dramatic increase in the number of neurons in dorsal root ganglia synthesizing the peptide cholecystokinin (CCK) after sciatic nerve section. Furthermore, the autotomy behavior of rats was significantly inhibited by chronic i.t. administration of morphine in conjunction with subcutaneous (s.c.) injection of CI 988. Neither i.t. morphine nor s.c. CI 988 alone produced a comparable effect on autotomy. Our results suggested that up-regulation of the mRNA for CCK in primary afferents after nerve injury may be related to the clinical phenomenon of opioid insensitivity. Thus, coadministration of CCK antagonists in combination with opioids may offer a new approach in treating neuropathic pain.

Evidence for endogenous inhibition of autotomy by galanin in the rat after sciatic nerve section: demonstrated by chronic intrathecal infusion of a high affinity galanin receptor antagonist.

We have studied the effect of M-35 [Galanin(1-12)-Pro-bradykinin(2-9)-amide], a newly developed high affinity antagonist for galanin receptors, on self-mutilation (autotomy) behavior of the deafferented limb in rats after unilateral section of sciatic nerves. M-35 (1.3 micrograms/microliters) or saline was applied to the lumbar spinal cord through a chronically implanted intrathecal catheter at a rate of 0.5 microliter/h for 10 days post axotomy via an osmotic minipump. Axotomized rats infused with M-35 autotomized significantly more than those perfused intrathecally with saline or those axotomized rats not implanted with an intrathecal catheter. The severity of autotomy was also markedly greater in the group treated with M-35 than in the two other groups. M-35 did not noticeably influence either the galanin mRNA level in corresponding dorsal root ganglia and dorsal horn region or the percent of lumbar sensory neurons expressing detectable levels of mRNA for galanin. It is suggested that galanin can endogenously suppress autotomy behavior in rats after nerve injury and thus may play an important role in the control of the development of neuropathic pain.