Selective Sensory Axon Reinnervation and TRPV1 Activation.

Current strategies to enhance regeneration of peripheral neurons involve broad activation of sensory, autonomic, and motor axons. Peripheral neuron regeneration is limited in persons with damage or disease of peripheral axons. Here, we provide evidence that subtoxic activation of TRPV1 channels in sensory neurons is associated with activation of growth and subtle changes in skin reinnervation. We identify a bidirectional, dose-related impact of capsaicin, a TRPV1 agonist, on sensory neurons and their axons with rises in their outgrowth plasticity at low doses and toxic neurodegeneration at high doses. Moreover, its impact on growth added to that of preconditioning. Neither outcome was observed in TRPV1 null neurons. We confirmed that low dose activation was associated with rises in neuronal calcium, as well as rises in TRPV1 mRNA transcripts. In mice with a sciatic nerve crush followed by a single application of capsaicin directly to the injury site, there was no impact on motor or myelinated axon recovery but there was evidence of better recovery of thermal sensation toward baseline with hyperalgesia. Moreover, skin reinnervation by epidermal axons approached contralateral levels. TRPV1 null mice displayed loss of thermal sensation during later recovery. In sensory axons innervating the pinna of the ear, local capsaicin rendered early axon loss followed by later hyperinnervation. Taken together, TRPV1 activation alters the regenerative behavior of adult neurons and their axons both in vitro and during epidermal reinnervation in vivo. The findings identify a selective manipulation that augments cutaneous innervation by thermosensitive axons.

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.

Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr.

Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF-responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1(-/-)) mice displayed allodynia (p<0.05), diminished epidermalinnervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1(-/-)) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin-related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor agonist indicated that NGFacted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr's effect on DRG neurons. Overall, NGF/TrkA signaling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.

Activated RHOA and peripheral axon regeneration.

The regeneration of adult peripheral neurons after transection is slow, incomplete and encumbered by severe barriers to proper regrowth. The role of RHOA GTPase has not been examined in this context. We examined the expression, activity and functional role of RHOA GTPase and its ROK effector, inhibitors of regeneration, during peripheral axon outgrowth. We used qRT-PCR, quantitative immunohistochemistry, and assays of RHOA activation to examine expression in sensory neurons of rats with sciatic transection injuries. In vitro, we exposed dissociated adult sensory neurons, not grown on inhibitory substrates, to a RHOA-ROK inhibitor HA-1077 and measured neurite initiation and outgrowth. In vivo, we exposed early regenerating axons and Schwann cells directly to HA-1077 in a conduit connecting the proximal and distal stumps of transected sciatic nerves. Intact adult dorsal root ganglia sensory neurons expressed RHOA and ROK 1 mRNAs and protein and there were rises in RHOA after injury. Activated GTP-bound RHOA, undetectable in intact ganglia, was dramatically upregulated in both neurons and axons after injury. Adult rat sensory neurons in vitro demonstrated a dose-related increase in the initiation of neurite outgrowth, and in the proportion with long neurites when they were exposed to a ROK antagonist. Regenerative bridges that were directly exposed to the ROK inhibitor had a dose-related rise in the extent and distance of in vivo axon and partnered Schwann cell regrowth within them. RHOA activation and signaling are features of adult peripheral axon regeneration within its own milieu, independent of myelin. Inhibition of its activation may benefit peripheral axon lesions.

Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance.

Growth cones use cues in their environment in order to grow in a directed fashion to their targets. In Xenopus laevis, fibroblast growth factors (FGFs) participate in retinal ganglion cell (RGC) axon guidance in vivo and in vitro. The main intracellular signaling cascades known to act downstream of the FGF receptor include the mitogen-activated protein kinase (MAPK), phospholipase Cgamma (PLCgamma) and phosphotidylinositol 3-kinase (PI3K) pathways. We used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF-2-stimulated RGC axon extension and chemorepulsion. The MAPK, PI3K and PLCgamma pathways were blocked by U0126, LY249002 and U73122, respectively. D609 was used to test a role for the phosphotidylcholine-PLC (PC-PLC) pathway. We determined that the MAPK and two PLC pathways are required for FGF-2 to stimulate RGC neurite extension in vitro, but the response of axons to FGF-2 applied asymmetrically to the growth cone depended only on the PLC pathways.

Fibroblast growth factors redirect retinal axons in vitro and in vivo.

Growth factors have been shown previously to participate in the process of axon target recognition. We showed that fibroblast growth factor receptor (FGFR) signaling is required for Xenopus laevis retinal ganglion cell (RGC) axons to recognize their major midbrain target, the optic tectum [neuron 17 (1996), 245]. Therefore, we have hypothesized that a change in expression of a fibroblast growth factor (FGF) at the entrance of the optic tectum, the border between the diencephalon and mesencephalon, may serve as a signal to RGC axons that they have reached their target. To determine whether RGC axons can sense changes in FGF levels, we asked whether they altered their behavior upon encountering an ectopic source of FGF. We found that in vivo RGC growth cones avoided FGF-misexpressing cells along their path, and that FGF-2 directly repelled RGC growth cones in an in vitro growth cone turning assay. These data support the idea that RGC axons can sense changes in FGF levels, and as such provide a mechanism by which FGFR signaling is involved in RGC axon target recognition.

Mucinous cystadenocarcinoma of the parotid gland: report of a case with fine needle aspiration findings and histologic correlation.

BACKGROUND: Mucinous cystadenocarcinoma of the salivary gland is a rare entity. Review of the literature from 1991 to 1999 revealed no previous reports on its cytologic features. CASE: A 25-year-old man had a slowly growing, painless mass in the left parotid gland. Fine needle aspiration biopsy, performed prior to surgical excision, showed clusters of minimally atypical epithelial cells in which occasional vacuolated cells containing mucin could be seen. Pathologic evaluation of the resected parotid mass showed it to be a mucinous cystadenocarcinoma. CONCLUSION: The cytologic differential diagnosis of mucinous cystadenocarcinoma is with low grade mucoepidermoid carcinoma and with mucinous adenocarcinoma. Mucinous cystadenocarcinoma must be cystic; cysts may be present in low grade mucoepidermoid carcinoma, but their size and prominence varies. Mucinous adenocarcinoma is not cystic but gelatinous. Nuclei are bland in both mucinous cystadenocarcinoma and low grade mucoepidermoid carcinoma but are atypical in mucinous adenocarcinoma. There is no squamous differentiation in either mucinous cystadenocarcinoma or mucinous adenocarcinoma, but it is subtle in low grade mucoepidermoid carcinomas. Mucinous cystadenocarcinoma should be considered a potential candidate in the differential diagnosis of mucinous lesions that can occur in the salivary gland.

Cyclic AMP prevents an increase in GAP-43 but promotes neurite growth in cultured adult rat dorsal root ganglion neurons.

High expression of the growth-associated protein GAP-43 in neurons is correlated with developmental and regenerative axon growth. It has been postulated that during development and after injury, GAP-43 expression is elevated due to the unavailability of a target-derived repressive signal, but that GAP-43 expression then declines upon target contact. Here we examine the cyclic AMP second messenger signaling pathway to determine if it might mediate retrograde transmission of a signal which represses GAP-43 expression and inhibits growth. Cultures of adult rat dorsal root ganglia were chronically exposed to membrane-permeable analogs of cyclic AMP and activators of adenyl cyclase. These treatments caused GAP-43 protein levels to decrease in a dose-dependent manner, although neuronal survival was not affected. GAP-43 mRNA was also decreases by cyclic AMP. GAP-43 protein levels were not repressed by neurotrophins, cytokines, or other agents. Surprisingly, cyclic AMP caused an increase in the rate of neurite outgrowth, even though the neurons were partially depleted of GAP-43. Growth stimulation was quickly inducible and reversible, could occur in the presence of transcription inhibitors, and did not entail alterations in branching pattern. These findings suggest that axon growth involving high levels of GAP-43 is distinct from the growth stimulation which is rapidly induced by cyclic AMP.

Divergent regulation of GAP-43 expression and CNS neurite outgrowth by cyclic AMP.

Robust process outgrowth and high expression of the growth-associated protein GAP-43 seem to be intrinsic features of neurons, but both are down-regulated after axonal contact of target cells. We report that chronic exposure of the serotonergic CNS cell line RN46A to cyclic AMP analogs, forskolin, or cholera toxin represses GAP-43 expression in a dose dependent manner. Thus, cAMP could mediate a GAP-43 repressive signal that is initiated extracellularly. Activation of the cyclic AMP pathway by these same reagents, however, enhances the rate that RN46A cells extend neurites. This stimulation of neurite growth can occur during inhibition of new transcription, and in the absence of high levels of GAP-43. These findings demonstrate that a GAP-43-repressing intracellular signaling pathway exists, that repression of GAP-43 expression by cAMP is not directly coupled to inhibition of neurite growth, and that acceleration of growth cone advancement by cAMP is not dependent on the presence of GAP-43.

Involvement of the amino-terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli.

The UhpA protein is required for expression of the sugar phosphate transporter UhpT in Escherichia coli and is regulated by phosphate transfer from the transmembrane UhpBC sensor kinase complex. UhpA action requires the sensor kinase complex and the site of phosphorylation, Asp-54, under normal conditions, but not when UhpA is overexpressed. Directed mutagenesis of the uhpA gene allowed examination of the role of several residues of UhpA in response to phosphorylation and in transcription activation. Residues Asp-9, Asp-54, and Lys-101 are highly conserved and required for function in other response regulators. Changes at any of these residues in UhpA resulted in complete loss of phosphorylation-dependent activity, but did not affect the high-level, constitutive, UhpBC-independent expression when the UhpA variants were overexpressed. Thus, these residues are important for the response to the phosphorylation pathway but not for transcription activation. Eight independent uhpA mutants selected for activity in the absence of UhpBC function carried the F17-->V or H170-->Y substitutions. Other substitutions for Phe-17 conferred various phenotypes, ranging from inducible to high-level constitutive behaviour. Residues in helix-1 flanking Phe-17 were converted to Ala or other residues. Alanine substitutions at Val-13, Arg-14, and Leu-20 resulted in complete loss of phosphorylation-dependent activation. Change of Gly-16 to Ala had no effect, but changes to other residues resulted in loss of function. Alanine substitutions at Phe-17 and at Gln-19 resulted in high-level constitutive expression, and changes at Ala-18 and Leu-21 had only modest effects. Most interesting was the L20-->A substitution, which conferred low uhpT expression when overexpressed and interfered with action of the wild-type chromosomal allele. The combination of the L20-->A change with changes at Phe-17, Asp-54 and His-170 indicated that the trans-dominant action of L20-->A occurred at several steps. The observations that UhpA can activate uhpT transcription in its unphosphorylated state are consistent with its occupancy of low-affinity binding sites necessary for promoter function. We propose that the effect of phosphorylation of UhpA is to enhance its oligomerization on the DNA surface to extend to the low-affinity sites, and that helix-1 participates in the process of oligomer formation.

Action of receiver and activator modules of UhpA in transcriptional control of the Escherichia coli sugar phosphate transport system.

Induction of the sugar-phosphate transport system in Escherichia coli by external glucose-6-phosphate is regulated by the UhpABC regulatory proteins. UhpA protein is required for uhpT transcription and is related to response regulators of two-component regulatory systems. UhpA and its homologues appear to be composed of two modules: the receiver module which contains the putative site of phosphorylation, and the activation module whose predicted helix-turn-helix motif is related to that present in many transcription activators. The roles of the two modules were examined by analysis of the regulatory consequences of uhpA deletion mutations generated by in vitro manipulations and missense mutations selected for independence from the requirement for UhpB kinase activity. Deletion of even seven amino acids from the C-terminus resulted in complete loss of transcription activation at the uhpT promoter. Overexpression of all C-terminal truncations that left intact the receiver module (residues 1-120) exhibited strong dominant-negative interference with a chromosomal uhpA+ allele. The genetic requirements for interference indicated that the overexpressed receiver module competed with intact UhpA for phosphate residues carried on UhpB. The site of phosphorylation of UhpA is not necessary for uhp activation by overexpressed UhpA but is necessary for UhpA action at normal levels of UhpA or for interference by the truncated species.

The adequacy of cytology and colposcopy in diagnosing cervical neoplasia in HIV-seropositive women.

The purpose of this study was to compare cytology and colposcopy as predictors of cervical intraepithelial neoplasia (CIN) in women infected with the human immunodeficiency virus (HIV). A cross-sectional analysis of cytology, colposcopy, and colposcopic biopsy results from 51 HIV-seropositive women attending an ambulatory HIV service was conducted. Cytology slides were reviewed by two cytopathologists blinded to patients' HIV status. There was strong agreement in the readings of two cytopathologists, with a kappa score of 0.9. Of 29 women with normal cytology, 21 (72%) had pathology on histology, including 7 (24%) with CIN. Colposcopic impression correlated well with histology results. Of 22 women with abnormal cytology, 82% had abnormal histology. The overall prevalence of CIN was high at 45%, increasing from 35% in women with CD4 counts over 400 to 56% in women with CD4 counts below 200. In conclusion, screening cytology is limited by false-negative results; routine colposcopy should be considered in this high-risk population.

Characteristics of cervical intraepithelial neoplasia in women infected with the human immunodeficiency virus.

OBJECTIVE: Our purpose was to compare characteristics of cervical intraepithelial neoplasia in relation to human immunodeficiency virus infection among women referred to a public hospital colposcopy clinic with Papanicolaou smears showing cervical intraepithelial neoplasia. STUDY DESIGN: An evaluation of cervical intraepithelial neoplasia severity, lesion size, and vulvovaginal lesions with respect to human immunodeficiency virus status was performed. RESULTS: (1) Human immunodeficiency virus prevalence in 482 women with cytologic characteristics of cervical intraepithelial neoplasia was 13%. (2) In human immunodeficiency virus-infected patients, Papanicolaou smears were less adequate for evaluation and correlated less well with histologic findings than in uninfected patients (p < 0.05). (3) Human immunodeficiency virus-infected patients (n = 47) had more advanced cervical intraepithelial neoplasia, larger cervical lesions, and more associated vulvovaginal lesions than human immunodeficiency virus-negative patients (n = 161). In human immunodeficiency virus-positive women, the severity of cervical intraepithelial neoplasia was not associated with age, whereas in human immunodeficiency virus-negative women, increasing severity was significantly associated with increasing age. CONCLUSION: High rates of human immunodeficiency virus infection in inner-city colposcopy services and high-grade, extensive cervical lesions in infected women warrant special attention.

The family of organo-phosphate transport proteins includes a transmembrane regulatory protein.

This review article briefly summarizes aspects of our current understanding of the Uhp sugar phosphate transport system in enteric bacteria, particularly the mode of genetic regulation of its synthesis. This regulation occurs by a process that involves an example of the very widespread and ever-growing group of so-called two-component bacterial regulatory systems, a mechanism of response to environmental signals that employs phosphate transfer reactions between constituent proteins. Of emphasis here is the unusual involvement in transmembrane signaling of the UhpC protein which is related in sequence and structure to some transport proteins, including the very protein whose synthesis it helps regulate.

Pap smear histories in a medical clinic: accuracy of patients' self-reports.

Women using the medical clinic of a public hospital were interviewed about their Pap smear histories to assess the accuracy of self-reported smears and to identify groups in need of further screening. Interview data from 263 women were compared with cytology files and hospital records. In spite of considerable agreement between patient report and record, patients reported significantly more recent smears than were documented. Accuracy of recall was not dependent on age or birthplace, but on the length of time since the last smear. About half the women had been screened within the past two years, whereas one tenth had never been screened. Women aged 65 years or older had fewer recent smears than younger women, while foreign born women were more likely never to have had a Pap smear than were United States born women. We conclude that self-reported information is useful in assessing Pap smear histories, but the screening rates that result should be treated as high estimates. Significant opportunities for screening exist in ambulatory care sites in low-income communities.

Bronchitis mimicking opportunistic lung infection in patients with human immunodeficiency virus infection/AIDS.

Purulent bronchitis was identified in 19 of 422 patients undergoing fiberoptic bronchoscopy during a 32-month period because of suspicion of an opportunistic lung infection complicating acquired immunodeficiency syndrome or human immunodeficiency virus infection. Five patients had Pneumocystis carinii pneumonia, but other opportunistic lung infections were excluded in the remaining 14 patients. Characteristics of these 14 patients included fever (greater than 38.3 degrees C), cough, and dyspnea in 14 of 14 patients; purulence of expectorated sputum (11/14); and widened alveolar-arterial oxygen gradient (13/14). Rapid (2 +/- 1.4 days) clinical response (defervescence and resolution of pulmonary symptoms) occurred with antibiotic therapy in 10 of 14 patients. In three patients, there was no improvement, and adult respiratory distress syndrome developed. Bacterial isolates from bronchoalveolar lavage included Streptococcus viridans (n = 12), Haemophilus influenzae (n = 7), Staphylococcus aureus (n = 3). Roentgenographic features of bronchiectasis were present in seven patients. Differential cell counts revealed greater than 50% neutrophils in the bronchial washings of all patients with purulent bronchitis. Neutrophil percentages in bronchoalveolar lavage were as follows: patient with purulent bronchitis without P carinii pneumonia (n = 14), 54.53% +/- 29.18%; patients with purulent bronchitis and concomitant P carinii pneumonia (n = 5), 62% +/- 31.9%. In a control group of 17 patients with P carinii pneumonia who did not have purulent bronchitis, the neutrophil percentage was 6.8% +/- 6.17% (p = less than 0.00001, t-test). Purulent bronchitis appears to be a distinct, treatable entity in patients with HIV infection and may accompany bacterial pneumonia, bronchiectasis, and P carinii pneumonia.

Papanicolaou smear abnormalities in ambulatory care sites for women infected with the human immunodeficiency virus.

OBJECTIVE: We attempted to determine the prevalence of cervical dysplasia in human immunodeficiency virus-infected women in ambulatory care settings and to correlate Papanicolaou smears with demographic and clinical variables. STUDY DESIGN: Papanicolaou smears of 135 women attending three ambulatory care clinics were reviewed. Chart review identified demographic and clinical variables, including CD4 count. Prevalence of abnormal smears was compared with baseline community rates. Demographic and clinical variables were correlated with Papanicolaou results with the chi 2 test. RESULTS: Fivefold to eightfold increased rates of abnormal smears in human immunodeficiency virus-infected women were observed. Prevalence of abnormal smears increased from 21% in women with CD4 counts greater than 600/mm3 to 45% in women with CD4 counts less than 400/mm3. Age, ethnicity, or mode of human immunodeficiency virus transmission was not significantly correlated with Papanicolaou smear findings. CONCLUSION: Increased rates of abnormal Papanicolaou smears and significant correlation with CD4 counts were observed in human immunodeficiency virus-infected women at ambulatory care sites. We recommend comprehensive gynecologic care, including semiannual Papanicolaou smears, for all human immunodeficiency virus-infected women.

Nonbronchoscopic lung lavage for diagnosis of opportunistic infection in AIDS.

Thirty patients known to have or suspected of having acquired immunodeficiency syndrome (AIDS) were evaluated for opportunistic pulmonary infection using a double lumen lavage catheter (DLL). Lavage specimens obtained were cytocentrifuged and initially stained by the Papanicolaou technique as a means of rapid evaluation for Pneumocystis carinii. If no opportunistic organism was identified, the patient underwent further diagnostic investigations. In 18 patients receiving mechanical ventilatory support, the procedure was performed via the endotracheal tube. Twelve patients who were less severely ill underwent the procedure via the transnasal route. In 43 percent (13/30), opportunistic infections were diagnosed by DLL. Twelve had P carinii, one of whom had cytomegalovirus and another of whom had Herpes simplex viruses, and one with Toxoplasma gondii. Thus, the sensitivity for all opportunistic infections was 86 percent (12/14). The volume of fluid recovered averaged 93 percent of that instilled. There was no significant difference between prelavage and postlavage PaO2. In this group of patients, double lumen lavage obviated the need for more invasive and expensive procedures.

Value of bronchoalveolar lavage in the diagnosis of pulmonary infection in acquired immune deficiency syndrome.

Seventy five patients with pulmonary disease and suspected acquired immune deficiency syndrome (AIDS) underwent fibreoptic bronchoscopy with bronchoalveolar lavage. Of 54 cases of Pneumocystis carinii pneumonia, 53 (98%) were diagnosed by bronchoalveolar lavage. Complications were recorded in 12 instances and included pneumothorax in two and transient increase in fever and hypoxaemia in the remainder. Bronchoalveolar lavage is a safe, easy, and effective procedure for diagnosing pneumocystis pneumonia in patients at high risk of AIDS and should be done routinely when fibreoptic bronchoscopy is performed in such patients.