Macrophage response to peripheral nerve injury: the quantitative contribution of resident and hematogenous macrophages

Whereas local microglial cells of the CNS rapidly respond to injury, little is known about the functional role of resident macrophages of the peripheral nervous system in nerve pathology. Using bone marrow chimeric rats, we recently identified individual resident endoneurial macrophages that rapidly became activated after nerve injury. However, the extent of local macrophage activation and its quantitative contribution to the total macrophage response is unknown. We now have created chimeric mice by transplanting bone marrow from green fluorescent protein (GFP)-transgenic mice into irradiated wild-type mice, allowing easy differentiation and quantification of hematogenous and resident endoneurial macrophages. After sciatic nerve crush injury, both GFP(-) and GFP(+) resident macrophages, the latter having undergone physiological turnover from the blood before injury, rapidly underwent morphological alterations and increased in number. Proliferating GFP(-) and GFP(+) resident macrophages were abundant and peaked 3 days after injury. A major lesion-induced influx of hematogenous macrophages with a disproportionate increase of GFP(+) macrophages was not observed until Day 4. Throughout all time points examined, GFP(-) resident macrophages were strikingly frequent, reaching maximum numbers 9.5-fold above baseline. There was also a notable proportion of GFP(-) resident endoneurial macrophages phagocytosing myelin and expressing major histocompatibility complex class II. Our results demonstrate for the first time that the rapid response of resident endoneurial macrophages to nerve injury is quantitatively important and that local macrophages contribute significantly to the total endoneurial macrophage pool during Wallerian degeneration.     

Subsynaptosomal distribution of calcium during aging and 3,4-diaminopyridine treatment

Since previous studies showed that calcium uptake by synaptosomes from rodents declines with aging [30], the subsynaptosomal distribution of calcium was determined with the disruption method of Scott et al. [37]. Calcium uptake by the mitochondrial (digitonin-resistant) and non-mitochondrial (digitonin-labile) compartments, as well as total uptake, were determined at 2, 5 and 10 min. After a 10 min incubation under resting conditions (5 mM-KCl), total calcium uptake decreased at 10 months (−14.6%) and 30 months (−33.0%) of age; mitochondrial calcium uptake increased by 10 months (+11.2%) but declined by 30 months (−17.5%); the nonmitochondrial calcium compartment declined at 10 (−34.7%) and 30 (−43.4%) months when compared to the 3 month old control. With potassium depolarization (31 mM-KCl), total calcium uptake declined from 100% (3 months) to 73.8% (10 months) or 53.0% (30 months); mitochondrial calcium uptake declined from 100% (3 months) to 85.6% (10 months) or 68.4% (30 months); non-mitochondrial calcium uptake decreased at 10 (−34.3%) and 30 (−57.7%) months of age when compared to 3 months (100%). The deficits in calcium homeostasis are not due to changes in synaptosomal volumes or to diminished membrane potentials, as assessed by tetraphenylphosphonium ion accumulation. 3,4-Diaminopyridine partially reversed the alterations in total, mitochondrial and non-mitochondrial calcium uptake by synaptosomes from aged mice.     

Glucose determination in human aqueous humor with Raman spectroscopy

It has been suggested that spectroscopic analysis of the aqueous humor of the eye could be used to indirectly predict blood glucose levels in diabetics noninvasively. We have been investigating this potential using Raman spectroscopy in combination with partial least squares (PLS) analysis. We have determined that glucose at clinically relevant concentrations can be accurately predicted in human aqueous humor in vitro using a PLS model based on artificial aqueous humor. We have further determined that with proper instrument design, the light energy necessary to achieve clinically acceptable prediction of glucose does not damage the retinas of rabbits and can be delivered at powers below internationally acceptable safety limits. Herein we summarize our current results and address our strategies to improve instrument design.     

Detection of Parkin (PARK2) and DJ1 (PARK7) mutations in early-onset Parkinson disease: Parkin mutation frequency depends on ethnic origin of patients

Mutations in the Parkin (PARK2) and the DJ1 (PARK7) gene cause early-onset Parkinson disease (EOPD). We tested 75 Serbian EOPD patients for mutations in both genes by conventional mutational screening (SSCP/dHPLC/sequencing) to detect small sequence alterations and by gene dosage studies (quantitative PCR) to reveal deletions or multiplications of one or more exons. A compound heterozygous Parkin mutation (exon deletion and point mutation; [c.836_972del]+[c.1411C>T]; +1 is first nucleotide of GenBank AB009973.1) was identified in a patient who showed a relatively benign course after a disease onset at 41 years. Another case had a heterozygous exon deletion in DJ1 ([c.253_322del]+[?]) and presented with an age at onset of 45 years and a rapid disease course. In conclusion, Parkin mutations are surprisingly rare in our Serbian EOPD sample, suggesting that the mutation rate depends on the ethnic origin of the patients. Although DJ1 mutations appear to be rare, we confirm their role in EOPD and demonstrate the importance of gene dosage studies.     

Human cytomegalovirus UL144 gene polymorphisms in congenital infections

The human cytomegalovirus (HCMV) UL144 gene is a tumor necrosis factor-like receptor with the potential to affect HCMV virulence. HCMV strains display genetic variability in the UL144 region, and the analysis of a potential link between UL144 gene polymorphisms and disease severity has scarcely been studied. However, a correlation between the UL144 genotype and congenital-disease outcome has been reported in one previous study, with the observation that all asymptomatic infants had a single UL144 genotype. In order to confirm or refute this finding, we determined the UL144 polymorphisms of HCMV strains recovered from the amniotic fluids of 38 infected fetuses and compared them to HCMV strains obtained from 30 viremic adult controls. The UL144 sequences were distributed among five genotypes (A, B, C, AC, and AB), as previously described. We observed similar percentages of the three major genotypes A (37%), B (33%), and C (27%) in our population. The UL144 genotype distributions were similar among the group of infected adults and the group of infected fetuses and among symptomatic and asymptomatic fetuses (P < 0.05). In our series, all five UL144 genotypes could be vertically transmitted from mothers to fetuses, and all could cause symptomatic congenital infection. We concluded that determination of UL144 polymorphisms in cases of congenital infection is not relevant, since it is unlikely to help predict the outcome of the infection.     

ETV6/RUNX1 fusion at diagnosis and relapse: some prognostic indications

This study was undertaken in order to compare the interphase and metaphase cytogenetics of 28 patients with ETV6/RUNX1 positive acute lymphoblastic leukemia, at diagnosis and relapse. The median time to relapse was 26 months. The significant fusion positive population heterogeneity revealed at interphase by a commercial probe for ETV6/RUNX1 fusion has not been described before. Six diagnostic samples had a single abnormal population; others had up to five each, which differed in the numbers of RUNX1 signals, and in the retention or loss of the second ETV6 signal. In contrast, the number of fusion signals was more constant. At relapse, there were fewer populations; the largest or unique clone was sometimes a re-emergence of a minor, diagnostic one, with a retained copy of ETV6 and the most RUNX1 signals. Abnormal, fusion negative clones were identified in bone marrow samples at extra-medullary relapse. Variant three or four-way translocations, which involved chromosomes 12 and 21, were prominent among the complex rearrangements revealed by metaphase FISH. The frequency of their occurrence at diagnosis and reappearance at relapse, sometimes accompanied by minor clonal evolution, was another new observation. Other recurrent cytogenetic features included a second copy of the fusion signal in six cases, partial duplication of the long arm of the X chromosome in two cases, and trisomy 10 in three cases. In comparing our data with previously reported cases, a picture is beginning to emerge of certain diagnostic features, which may provide circumstantial evidence of an increased risk of relapse.     

Superinfecting mycobacteria home to established tuberculous granulomas

A central paradox of tuberculosis immunity is that reinfection and bacterial persistence occur despite vigorous host immune responses concentrated in granulomas, which are organized structures that form in response to infection. Prevailing models attribute reinfection and persistence to bacterial avoidance of host immunity via establishment of infection outside primary granulomas. Alternatively, persistence is attributed to a gradual bacterial adaptation to evolving host immune responses. We show here that superinfecting Mycobacterium marinum traffic rapidly into preexisting granulomas, including their caseous (necrotic) centers, through specific mycobacterium-directed and host cell-mediated processes, yet adapt quickly to persist long term therein. These findings demonstrate a failure of established granulomas, concentrated foci of activated macrophages and antigen-specific immune effector cells, to eradicate newly deposited mycobacteria not previously exposed to host responses.     

Focal expression and final activity of matrix metalloproteinases may explain irregular dysfunctional endometrial bleeding

Irregular dysfunctional bleeding of the endometrium (ie, metrorrhagia without organic lesion) is common in women, whether treated or not with ovarian hormones. Several matrix metalloproteinases (MMPs) become normally expressed and/or activated at menstruation and cause extracellular matrix breakdown. We therefore explored whether episodes of irregular dysfunctional bleeding could be associated with untimely MMP activity. By histology, foci of stromal breakdown were exclusively found in the endometrium of metrorrhagic women at bleeding. In these foci, 1) expression of estrogen receptor-alpha and progesterone receptor was altered; 2) collagenase-1 (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) became detected in stromal cells, together with MMP-9 in neutrophils; and 3) gelatinase A (MMP-2) was more expressed and immunolocalized at the membrane of stromal cells. By biochemistry, endometrial lysates from nonbleeding metrorrhagic patients contained more latent and active MMP-2 and -9 than age-matched controls; at bleeding, collagenase activity, MMP-9, and active MMP-2 were strikingly increased whereas tissue inhibitor of metalloproteinases-1 (TIMP-1) was considerably decreased. As a functional assay, in situ gelatin zymography revealed large areas of gelatinolytic activity only in endometrium of bleeding patients. Altogether, these results strongly suggest that inappropriate focal expression and activation of several MMPs, combined with decreased inhibition, trigger irregular dysfunctional endometrial bleeding.