Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to 276 Campylobacter jejuni strains and 87 Campylobacter coli strains isolated from humans, pigs, cattle, poultry, and retail meats to investigate whether certain FAFLP genotypes of C. jejuni and C. coli are associated with a particular host and to determine the degree of association between FAFLP-defined genotypes and heat-stable serotypes and/or phage types. Within C. coli, the poultry strains clustered separately from those of porcine origin. In contrast, no evidence of host specificity was detected among C. jejuni strains. While C. coli strains show host specificity by FAFLP genotyping, C. jejuni strains that are genotypically similar appear to colonize a range of hosts, rather than being host adapted. Some serotypes and/or phage types (C. jejuni serotype HS18, phage type PT6, and serophage type HS19/PT2 and C. coli HS66, PT2, and HS56/PT2) were the most homogeneous by FAFLP genotyping, while others were more heterogeneous (C. jejuni HS5 and PT39, and C. coli HS24 and PT44) and therefore poor indicators of genetic relatedness between strains. The lack of host specificity in C. jejuni suggests that tracing the source of infection during epidemiological investigations will continue to be difficult. The lack of congruence between some serotypes and/or phage types and FAFLP genotype underlines the need for phenotypic testing to be supplemented by genotyping. This study also demonstrates how, in general, FAFLP generates "anonymous" genetic markers for strain characterization and epidemiological investigation of Campylobacter in the food chain.     

Mitochondrial Abnormality Associates with Type-Specific Neuronal Loss and Cell Morphology Changes in the Pedunculopontine Nucleus in Parkinson Disease

Cholinergic neuronal loss in the pedunculopontine nucleus (PPN) associates with abnormal functions, including certain motor and nonmotor symptoms. This realization has led to low-frequency stimulation of the PPN for treating patients with Parkinson disease (PD) who are refractory to other treatment modalities. However, the molecular mechanisms underlying PPN neuronal loss and the therapeutic substrate for the clinical benefits following PPN stimulation remain poorly characterized, hampering progress toward designing more efficient therapies aimed at restoring the PPN''s normal functions during progressive parkinsonism. Here, we investigated postmortem pathological changes in the PPN of PD cases. Our study detected a loss of neurons producing gamma-aminobutyric acid (GABA) as their output and glycinergic neurons, along with the pronounced loss of cholinergic neurons. These losses were accompanied by altered somatic cell size that affected the remaining neurons of all neuronal subtypes studied here. Because studies showed that mitochondrial dysfunction exists in sporadic PD and in PD animal models, we investigated whether altered mitochondrial composition exists in the PPN. A significant up-regulation of several mitochondrial proteins was seen in GABAergic and glycinergic neurons; however, cholinergic neurons indicated down-regulation of the same proteins. Our findings suggest an imbalance in the activity of key neuronal subgroups of the PPN in PD, potentially because of abnormal inhibitory activity and altered cholinergic outflow.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Patients with Parkinson disease (PD) present with a multitude of motor-related disabilities, including progressive resting tremor, rigidity, bradykinesia/akinesia, gait disturbances, and postural instability. In addition, it is recognized increasingly that various nonmotor functions are also left impaired, including mood, cognition, sleep, autonomic nervous system functions, and sensory functions.¹ A neuropathological signature of PD is the progressive deterioration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc).² Although the precise cellular and molecular mechanisms underlying this neuronal death remain unknown, several reports implicate an underlying mitochondrial dysfunction, relating to energy deficits, enhanced production of free-radical species with concomitant oxidative stress,³ proteasomal deregulation,⁴ and neuronal excitotoxicity.⁵Evidence for a mitochondrial-related cause in PD stems from studies reporting on the use of human postmortem brains of patients with PD, which found a deficiency of complex I of the mitochondrial respiratory chain in the SNpc.⁶ Furthermore, outside the central nervous system a mitochondrial respiratory chain complex I deficiency has also been detected in the blood platelets of patients with PD, with some patients who also displayed defects of mitochondrial respiratory chain complexes II and III.⁷ In this regard, Gu et al⁸ found that a mitochondrial DNA (mtDNA) abnormality may underlie this mitochondrial defect in at least a proportion of patients with PD. By contrast, data reporting on mitochondrial respiratory chain defects in skeletal muscle cells of patients with PD remain somewhat more controversial. In this regard, Penn et al⁹ performed 31P magnetic resonance spectroscopy on the resting muscles of patients with PD, to report detecting defects in oxidative phosphorylation in the patients'' musculature, compared with healthy control cases. However, a study by Taylor et al¹⁰ was unable to validate this result. It has been proposed that the conflicting results that report on skeletal mitochondrial defects in patients with PD may relate to either methodological variation for assessing this biochemical defect or may be a reflection of the heterogeneity of the disease.¹¹Further evidence for an association between PD and a mitochondrial defect was obtained from the use of experimental neurotoxins such as rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Use of such toxins mimic parkinsonism in animals to a remarkably accurate extent, with studies showing that the pathological substrate for this defect may be due to the ability of such toxins to inhibit complex I of the mitochondrial respiratory chain.^12,13 Finally, disordered mitochondrial function, including defects in oxidative phosphorylation, are also seen in rare, young-onset genetic forms of PD, such as in patients who harbor mutations in genes such as Parkinson protein 2, E3 ubiquitin protein ligase [parkin (official symbol, PARK2)], parkinson protein 7 [DJ-1, (official symbol, PARK7)], and PTEN (phosphatase and tensin homologue) induced putative kinase 1 (PINK1), where loss of function of the respective protein products associate with deregulation of the mitochondrial quality control pathways of the cells.¹⁴Although the principal motor features of PD stem from reduced dopaminergic innervation of the striatum because of a substantial loss of SNpc dopaminergic neurons, recognition is growing that PD symptoms could result from disruption to multiple neural regions and systems.¹⁵ Although the loss of neurons is most conspicuous in the SNpc, neuronal loss and the presence of intracytoplasmic Lewy bodies (LBs) and Lewy neurites (LNs), composed of aggregation-prone proteins such as α-synuclein (αSYN) form an additional neuropathological hallmark of PD¹⁶ and have been observed in brain regions as diverse as the dorsal motor nucleus of vagus of the medulla, the locus ceruleus in the pons, the raphe nucleus, the basal forebrain, and allocortical regions such as the hippocampus and amygdala.¹⁷ Such widespread distribution of PD pathology could correlate with the variety of motor and nonmotor symptoms observed in patients with PD.¹⁸ PD-related pathologies that affect regions other than the dopaminergic-rich SNpc suggest that, although treatments that target only the nigrostriatal dopaminergic system could substantially benefit patients with PD, they are unlikely to completely resolve the PD-related deficit.¹⁹One particular brain region, the pedunculopontine nucleus (PPN), located within the lateral tegmental region and spanning the pontine midbrain isthmus, has been deemed critically important for regulating some of the physiological functions that fail during progressive PD. Such functions include regulating the activity of the reticular activating system for controlling rapid eye movement (REM) sleep.²⁰ Interestingly, patients with PD frequently present with abnormal REM muscle tone and concomitant REM sleep behavior disorder (RBD),²¹ which may be due to a loss of PPN cells and their concomitant functions during progressive PD. Moreover, PPN axons project toward and receive input from a variety of brain regions, including the thalamus, SN (both the compacta and reticular part), cortical regions, and spinal cord, all of which are involved in regulating aspects of voluntary motor function.^22–30 The PPN was assigned a role in the onset and progression of PD because of reports that the nucleus undergoes degenerative changes, principally affecting the resident cholinergic neurons.^31,32 The loss of these cells is believed to provide the cellular basis for the gait and postural deficits that patients with PD experience³³ and nonhuman primates rendered parkinsonian via cytotoxic lesions.^34,35 In addition, LBs and LNs are seen within the remaining PPN neurons in the postmortem brains of patients with PD.³⁶ Such findings provided the rationale for the commencement of therapeutic trials of deep brain stimulation of the PPN, with trial results reporting a reduction in gait and postural dysfunction in patients with PD after receiving PPN deep brain stimulation.^37,38Here, we used serially cut sections taken from the postmortem PPN of patients with PD and compared this with elderly, healthy control persons, who died without known neurological or psychiatric deficit. After confirming a decreased number of cholinergic neurons in PD-affected PPNs, as previously reported,^31,32 we studied whether the remaining cholinergic neurons undergo somatic cell size alterations. Because it is unknown whether other, noncholinergic neurons are also lost in the PPN of patients with PD, we next determined whether glycinergic and GABAergic neurons in the PPN also degenerate as a result of PD, and whether the remaining neurons undergo structural alterations. In an attempt to explain the altered cell numbers and cellular structural changes seen in the PPN of the present study''s cohort of patients with PD compared with controls, and consistent with the wide amount of literature that suggests an association between PD and mitochondrial dysfunction, we determined whether the loss of different neuronal subpopulations in the PPN is linked to mitochondrial abnormalities.     

Enhanced Pseudomonas aeruginosa biofilm development mediated by human neutrophils

Cystic fibrosis (CF) lung disease features persistent neutrophil accumulation to the airways from the time of infancy. CF children are frequently exposed to Pseudomonas aeruginosa, and by adulthood, 80% of CF patients are chronically infected. The formation of biofilms is a particularly important phenotypic characteristic of P. aeruginosa that allows for bacterial survival despite aggressive antibiotic therapy and an exuberant immune response. Here, we show that the presence of neutrophils enhances initial P. aeruginosa biofilm development over a period of 72 h through the formation of polymers comprised of actin and DNA. F-actin was found to be a site of attachment for P. aeruginosa. These actin and DNA polymers are present in CF sputum, and disruption of the polymers dispersed the associated P. aeruginosa cells and reduced biofilm development. These findings demonstrate a potential maladaptation of the primary innate response. When the host fails to eradicate the infection, cellular components from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation.     

Impaired mitochondrial beta-oxidation in a patient with an abnormality of the respiratory chain. Studies in skeletal muscle mitochondria.

Defects of complex I of the mitochondrial respiratory chain are important causes of neurological disease. We report studies that demonstrate a severe deficiency of complex I activity with less severe abnormalities of complexes III and IV (less than 5, 63, and 30% of control values, respectively) in a skeletal muscle mitochondrial fraction from a 22-yr-old female with weakness, lactic acidemia, and the deposition of intramuscular neutral lipid. The observation that lipid accumulates in this and other patients with complex I deficiency suggests impaired mitochondrial fatty acid oxidation. To investigate this mechanism we have shown impaired flux through beta-oxidation [( U-14C]hexadecanoate oxidation was 66% of control rate) and accumulation of specific acyl-CoA ester intermediates. The changes in fatty acid metabolism in complex I deficiency are secondary to the reduced state within the mitochondrial matrix with low NAD+/NADH ratios.     

Impact of a Palliative Care Consultation Team on Cancer-Related Symptoms in Advanced Cancer Patients Referred to an Outpatient Supportive Care Clinic

ContextPatients with advanced cancer may develop severe physical and psychosocial symptoms. There are limited data on the impact of an outpatient palliative consultation (PC) team on cancer-related symptoms.ObjectivesTo study the impact of the PC on symptoms in patients with advanced cancer receiving outpatient palliative care.MethodsFour hundred six consecutive patients referred to a supportive care outpatient center (OPC) from January 2006 to June 2007 with complete Edmonton Symptom Assessment Scale (0–10 scale) at the initial and follow-up visits were reviewed. Patient characteristics, change of symptoms at follow-up visit, and response rate were analyzed. Using logistic regression models, the predictors of improvement of pain and fatigue were assessed.ResultsMedian age was 59 years; 53% were female. Median interval between visits was 15 days. Mean scores at baseline and follow-up visits were fatigue 6.8 and 5.3 (P < 0.0001), pain 5.3 and 4.1 (P < 0.0001), depression 3.2 and 2.5 (P < 0.0001), anxiety 3.7 and 2.8 (P < 0.0001), dyspnea 2.7 and 2.5 (P = 0.05), sleep 5 and 4 (P < 0.0001), and well-being 5.2 and 4.4 (P < 0.0001). Dyspnea (odds ratio and P-value, 0.90, 0.03), nausea (0.92, 0.06), and depression (0.91, 0.04) were associated with improvement in fatigue; drowsiness (1.10, 0.04), and feeling of well-being (0.87, 0.02) were associated with improvement in pain.ConclusionThe initial consult by PC achieved significant symptom improvement in patients receiving treatment in the OPC. Further prospective studies are needed.     

Infectivity of Chlamydia trachomatis serovar LGV but not E is dependent on host cell heparan sulfate

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity of Chlamydia trachomatis serovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody to C. trachomatis by flow cytometry. Results showed no binding of the heparan sulfate antibody to C. trachomatis serovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.