CCL-1 in the spinal cord contributes to neuropathic pain induced by nerve injury.

Cytokines such as interleukins are known to be involved in the development of neuropathic pain through activation of neuroglia. However, the role of chemokine (C-C motif) ligand 1 (CCL-1), a well-characterized chemokine secreted by activated T cells, in the nociceptive transmission remains unclear. We found that CCL-1 was upregulated in the spinal dorsal horn after partial sciatic nerve ligation. Therefore, we examined actions of recombinant CCL-1 on behavioural pain score, synaptic transmission, glial cell function and cytokine production in the spinal dorsal horn. Here we show that CCL-1 is one of the key mediators involved in the development of neuropathic pain. Expression of CCL-1 mRNA was mainly detected in the ipsilateral dorsal root ganglion, and the expression of specific CCL-1 receptor CCR-8 was upregulated in the superficial dorsal horn. Increased expression of CCR-8 was observed not only in neurons but also in microglia and astrocytes in the ipsilateral side. Recombinant CCL-1 injected intrathecally (i.t.) to naive mice induced allodynia, which was prevented by the supplemental addition of N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801. Patch-clamp recordings from spinal cord slices revealed that application of CCL-1 transiently enhanced excitatory synaptic transmission in the substantia gelatinosa (lamina II). In the long term, i.t. injection of CCL-1 induced phosphorylation of NMDA receptor subunit, NR1 and NR2B, in the spinal cord. Injection of CCL-1 also upregulated mRNA level of glial cell markers and proinflammatory cytokines (IL-1ß, TNF-α and IL-6). The tactile allodynia induced by nerve ligation was attenuated by prophylactic and chronic administration of neutralizing antibody against CCL-1 and by knocking down of CCR-8. Our results indicate that CCL-1 is one of the key molecules in pathogenesis, and CCL-1/CCR-8 signaling system can be a potential target for drug development in the treatment for neuropathic pain.

Single air contrast arthrography for temporomandibular joint disorder using limited cone beam computed tomography for dental use.

MRI has the problem of artefacts caused by metal or body motion and is also incompatible for patients with claustrophobia. Arthrography is invasive and involves the risk of perforation or allergy to a contrast medium. This report discusses a patient with temporomandibular joint (TMJ) disorder who required soft tissue imaging of the TMJ. As she had claustrophobia and a reaction to iodine, air contrast arthrography and pumping manipulation therapy using limited cone beam computed tomography for dental use (3DX) was performed. We conclude that the 3DX examination method used in the study is practical as a diagnostic procedure and thus recommend this method to be used for patients with TMJ disorder in the presence of iodine contraindication.

Treatment of non-inflamed obstructive meibomian gland dysfunction by an infrared warm compression device.

AIM: To test the short term efficacy and safety of an infrared warm compression device (IWCD, Eye Hot, Cept Co, Tokyo, Japan) as treatment for non-inflamed meibomian gland dysfunction (MGD). METHODS: 37 subjects with non-inflamed obstructive MGD, with and without aqueous tear deficiency (ATD) dry eye, participated in a prospective non-comparative interventional case series. Symptom scores, face scores, tear evaporation rates, fluorescein and rose bengal vital staining, tear break up time (BUT), Schirmer test, meibomian gland obstruction, and meibography were compared before and after 2 weeks of therapy. RESULTS: In a total of 37 cases, total subjective symptom scores and subjective face scores improved significantly, from 12.3 (SD 5.9) to 8.4 (6.1), and from 7.0 (1.7) to 5.3 (2.0) (both p <0.0001). The results for tear evaporation rates during forced blinking (p = 0.002), fluorescein staining (p = 0.03), rose bengal staining (p = 0.03), BUT (p <0.0001), and meibomian gland orifice obstruction score (p <0.0001) had also improved significantly at the end of the 2 week period of infrared thermotherapy. No complaints and/or complications of the IWCD were reported. CONCLUSION: The IWCD was effective and safe for the treatment of MGD. Improved tear stability associated with release of meibum is a possible mechanism of this treatment.

Retinal dysfunction in cancer-associated retinopathy is improved by Ca(2+) antagonist administration and dark adaptation.

PURPOSE: It was recently found that recoverin acts as an autoantigen recognized by sera of patients with cancer-associated retinopathy (CAR), and that CAR-like retinal dysfunction is produced by intravitreous administration of anti-recoverin antibody in Lewis rat eyes. To examine the pathologic molecular mechanism of CAR, and to elucidate an effective therapy for CAR, the function and morphology of CAR were compared with those of phototoxic retinal damage, another form of photoreceptor dysfunction, and the effect of nilvadipine, a Ca(2+) antagonist, on the retinal degenerations was studied, using these models. METHODS: Under different illumination conditions and/or medication with nilvadipine, the functional and morphologic properties of the retinas were evaluated after intravitreous injection of anti-recoverin antibody into Lewis rat eyes (six rats, 12 eyes in each experimental condition), using electroretinogram (ERG), rhodopsin phosphorylation, and light microscopy. RESULTS: Anti-recoverin antibody administered into the vitreous of Lewis rat eyes induced a significant decrease and increase of ERG responses and rhodopsin phosphorylation levels, respectively, under cyclic or continuous light. Similar changes were observed in eyes of rats bred under continuous illumination that did not receive anti-recoverin antibodies. However, anti-recoverin antibody-induced retinal dysfunctions were not observed in rat eyes under dark conditions. Administration of nilvadipine, a Ca(2+) antagonist, to the anti-recoverin antibody-treated rats and rats with phototoxic retinal dysfunction caused significant improvement of the deterioration of ERG and normalization of rhodopsin phosphorylation. CONCLUSIONS: The present data indicate that anti-recoverin antibody-induced retinal dysfunction was functionally similar to phototoxic retinal dysfunction and was markedly suppressed under dark conditions or by systemic administration of a Ca(2+) antagonist.

Increases of calcium and magnesium and decreases of phosphorus and iron with aging in human uterine tubes.

To elucidate compositional changes of the uterine tube by aging, the authors studied age-related changes of elements in human uterine tubes by inductively coupled plasma-atomic emission spectrometry. The uterine tubes were resected postmortem or surgically removed from patients with uterine myoma. It was found that the contents of calcium and magnesium increased progressively with aging in uterine tubes, whereas the contents of phosphorus and iron decreased gradually with aging. The sulfur content of uterine tubes remained constant and independent of aging. Regarding relationships between elements, significant relationships were found between calcium and magnesium contents, between phosphorus and iron contents, between phosphorus and sulfur contents, and between phosphorus and sodium contents in human uterine tubes.

Novel fungal transcriptional activators, Cmr1p of Colletotrichum lagenarium and pig1p of Magnaporthe grisea, contain Cys2His2 zinc finger and Zn(II)2Cys6 binuclear cluster DNA-binding motifs and regulate transcription of melanin biosynthesis genes in a developmentally specific manner.

Colletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichum melanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA-binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1Delta mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.

Age-dependent changes of elements in human trachea.

To elucidate compositional changes of human trachea by aging, element contents in tracheae were determined by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The sulfur content of the tracheae decreased gradually with aging. In regard to calcium and phosphorus, both the contents increased to about threefold amounts in their seventies compared with those in their sixties, and decreased thereafter. The contents of calcium and phosphorus were the highest in their seventies. Therefore, it is likely that surplus calcium released from bones is deposited temporally in the trachea, and the deposits are released from the trachea at older age. Based on our results of human cartilages, there are two types in regard to calcium accumulation: The first type is that calcium accumulation occurs progressively with aging; the second one is that calcium accumulation becomes the highest in the seventies or eighties, and decreases thereafter. Therefore, the trachea belongs to the second type. Furthermore, the magnesium content remained constant through the age range.

Age-dependent decreases of phosphorus and magnesium in human Achilles' tendons.

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles' tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles' tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles' tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles' tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.

[Renocolic fistula: a case report].

A 78-year-old woman had disregarded pneumaturia since April 1998. In March 1999, computed tomography and barium enema were done to examine anemia and positive fecal occult blood, revealing a left renocolic fistula and bilateral renal stones. Intravenous pyelography revealed a left staghorn calculus, non-functioning kidney, and right partial staghorn calculus. Urinalysis showed pyuria and the culture grew Proteus vulgaris and Klebsiella oxytoca. Smear and culture of the urine were negative for acid-fast bacilli. In consideration of the patient's age and conservation of right renal function, right pyelolithotomy was performed first. Three weeks later, left nephrectomy and partial colectomy were done. The postoperative course was uneventful. A renocolic fistula is relatively rare and to our knowledge there have been 37 cases reported in Japan, including our case. Surgery is the main therapy and was performed in 31 patients. Among them, surgery was not curative in 1 and 5 died of postoperative complications. Thus, surgery is not safe in all cases. However, despite her age and bilateral renal dysfunction, our patient was successfully operated on.

Treatment with active vitamin D metabolites and concurrent treatments in the prevention of hip fractures: a retrospective study.

UNLABELLED: The purpose of this study was to determine the effect of treatment with active vitamin D metabolites and other concurrent medication on the prevention of hip fractures in elderly women. We inspected the medical records of the entire female population over 65 years of age on Sado Island, and followed a total of 11,377 women for a 3-year period. Of these, 1208 osteoporotic patients were treated with either 1,25-(OH)2D3 or 1 alpha-(OH)D3. The 765 patients who received the minimum effective dosage for more than 6 months made up the 'treatment group'. Nearly half these patients were also treated with either calcitonin or calcium. The 443 patients who received treatment with active vitamin D metabolites, but at a dosage or for a duration that did not meet the criteria for the treatment group, were deemed the 'ineffective group'. The remaining 10,169 women were the 'non-treatment group'. Fractures in the non-treatment group occurred at a rate of 39.8 fractures/10,000 person-years. The rate in the treatment group was 10.8, which was significantly lower (p = 0.039). Interestingly, the fracture rate after ceasing treatment was 52.1, which was significantly higher (p = 0.002) than the rate in patients receiving treatment. No statistical differences in the fracture rate were found between the ineffective, non-treatment and post-treatment groups. A reduction in the fracture rate was observed only in the treatment subgroup that did not also receive calcitonin (p = 0.042), and not in the subgroup that also received calcitonin therapy (p = 0.333). However, there was no statistical difference in the hip fracture rates between these two subgroups (p = 0.157) and the actual number of fractures was minimal (0 vs 2). Therefore, in this study, the advantage of treatment with active vitamin D alone over combined treatment with calcitonin seems to be marginal. IN CONCLUSION: (1) treatment with active vitamin D metabolites and with combined therapy may be marginally effective in preventing hip fractures, and (2) stopping the treatment clearly increases the risk of hip fractures.

Age-related changes of elements in the human articular disk of the temporomandibular joint.

To elucidate compositional changes of the articular disk (AD) of the temporomandibular joint (TMJ) by aging, elements of the ADs resected from 18 cadavers were determined by inductively coupled plasma atomic-emission spectrometry. It was found that calcium contents of ADs in TMJs increased progressively with aging, whereas the sulfur contents of the ADs decreased slightly with aging. Regarding the content of phosphorus, the contents increased progressively with aging. The study revealed that age-related changes of calcium contents in the ADs of TMJs were similar to those in women's pubic symphyses, but not those in intervertebral disks and menisci.

Age-related changes of mineral contents in the human aorta and internal thoracic artery.

To elucidate accumulations of minerals in the human aorta and internal thoracic artery, their relative contents (RCs) of minerals were analyzed by inductively coupled plasma atomic emission spectrometry. Aortas from 47 men and 24 women subjects were examined. The ages of these subjects ranged from newborn to 99 yr. After the age of 40 yr, RCs of calcium and phosphorus began to increase, and thereafter increased stepwise in the 50s and 70s. In the 70s, their accumulations were markedly increased. Internal thoracic arteries from 16 men and 7 women subjects were examined. These subjects ranged in age from 65-93 yr. It was found that all the RCs of calcium were low, <5.0 mg/g dry wt, and there was no age-dependent increase of calcium contents in internal thoracic arteries.

Prognostic significance of Ki-67 expression in transitional cell bladder carcinoma after radical cystectomy.

We evaluated the prognostic significance of the Ki-67 labeling index (Ki-67 LI) in 75 patients with transitional cell carcinoma of the bladder who underwent radical cystectomy. Immunohistochemical staining of archival material was performed by the streptavidin-biotin method. Univariate survival analysis showed that Ki-67 LI (p < 0.001), histologic grade (p < 0.05), tumor stage (p < 0.001) and the number of positive lymph nodes (p < 0.001) significantly correlated with prognosis. Multivariate survival analysis indicated that the Ki-67 LI (p < 0.05), histologic grade (p < 0.01), tumor stage (p < 0.01), presence of lymph node metastases (p < 0.05) and use of neo-adjuvant therapy (p < 0.05) had independent prognostic value. The Ki-67 LI is an independent prognostic factor for patients with transitional cell bladder cancer treated by radical cystectomy.

A histomorphometric study on effects of single and concurrent intermittent administration of human PTH (1-34) and bisphosphonate cimadronate on tibial metaphysis in ovariectomized rats.

This study compared the single administration of hPTH(1-34), bisphosphonate cimadronate (YM-175), and concurrent therapy of these two for restoration of lost bone mass in ovariectomized (OVX) rats. Animals were untreated for 4 weeks after surgery, and then injected s.c. with vehicle (OVX+V), hPTH(1-34) (30 micrograms/kg) (OVX+P), YM-175 (5 micrograms/kg) (OVX+Y), or a combination of these two (OVX+P+Y), 3 days a week, for 8 weeks, and sacrificed. Their proximal tibia were processed undecalcified for quantitative bone histomorphometry. Although OVX+Y showed a reduction of bone turnover compared to OVX+V, it failed to restore lost bone mass in OVX rats. In contrast, OVX+P exhibited a stimulation of bone formation and restored cancellous osteopenia due to OVX. OVX+P+Y also resulted a recovery of osteopenia, however, stimulation of bone formation by PTH was suppressed by YM-175.

Histochemical localization and X-ray microanalysis of calcium in the rat submandibular gland: demonstration of possible sites for microlith induction.

Rapidly frozen and freeze-substituted submandibular glands of young female rats were embedded in Epon and processed for histochemical demonstration of calcium with the glyoxal bis (2-hydroxyanil) (GBHA) staining method. GBHA staining of thick Epon sections revealed discrete calcium reactions of moderate intensity in practically every secretory granule but not in other compartments of the acinar cells. The saliva in the excretory duct was also reactive with GBHA and showed a drastic decrease in staining intensity toward the distal segments of excretory ducts with larger diameters. In addition, the duct saliva contained numerous tiny particles that were highly GBHA reactive. Stromal cells and cells lining the excretory duct were totally free of reactions. In the acinar cells, X-ray analysis detected distinct peaks for calcium in secretory granules and smaller ones in the Golgi apparatus, while they were undetectable in the rough surfaced endoplasmic reticulum (RER), implicating post-RER calcium loading in the secretory pathway. Electron-dense deposits in the duct saliva showed distinct peaks both for calcium and phosphorus, though these appeared in the acinar secretory granules and other cytoplasmic regions lacked phosphorus. Our observations thus demonstrated physiological calcium in the intra- as well as extracellular compartments of the submandibular gland, and further confirmed drastic changes in chemical composition along the synthetic and secretory pathways of the saliva, by both histochemical and X-ray microanalytical methods. GBHA staining of calcium combined with X-ray microanalysis is useful for an evaluation of the physiology and histo-pathological changes of the salivary glands associated with initial phases of microliths as well as sialoliths formation.

Synthesis and antitumor activity of poly(ethylene glycol)s linked to 5-fluorouracil via a urethane or urea bond.

In order to provide a macromolecular prodrug of 5-fluorouracil (5FU) with reduced side-effects and exhibiting strong antitumor activity, 5FU was covalently linked to poly(ethylene glycol) (PEG) via a urethane or urea bond. For the purpose of evaluating the release behavior of 5FU, the hydrolysis of the urethane or urea bond in the obtained conjugate of PEG-end capped with 5FU was investigated in vitro at 37 degrees C in aqueous solution media. The survival effect for the conjugate was assessed in vivo against p388 lymphocytic leukemia in female CDF1 mice by intraperitoneal (i.p.) transplantation/i.p. injection. The effects of a hydrophobic hexamethylene spacer group, the end group and the number n of ethylene oxide (EO) units in PEG on the release behavior of 5FU and the survival effect were investigated. The release rate of 5FU from the 5FU-terminated PEG conjugates via urethane or urea bond was very fast. However, it became slow with increasing n of EO units in PEG and was depressed by the introduction of hydrophobic spacer group. The 5FU-terminated PEG conjugates obtained exhibited significant survival effects against p388 leukemia mice i.p./i.p. Especially, the methoxy PEG (n = 113)/urethane/hexamethylene/urea/5FU conjugate showed the strongest survival effect among the synthesized 5FU-capped PEG conjugates via urethane or urea bond compared to free 5FU against p388 leukemia mice. These conjugates obtained did not display an acute toxicity even in high dose ranges.

The role of apoptosis in the response of cells and tumours to mild hyperthermia.

There is now abundant evidence that apoptosis, the cell death mechanism responsible for physiological deletion of cells, can be triggered by mild hyperthermia. However, the overall importance of this mode of death in heated tumours has not yet been established. In this light and electron microscopic study, apoptosis induced by 43 degrees C or 44 degrees C water bath heating for 30 min in a range of murine and human tumours growing in vitro and in four murine tumours growing as solid nodules in vivo, was identified on the basis of its characteristic morphology, and the amount present quantified. Apoptosis was found to play a variable role in the response of tumours to heating, with the lowest levels produced in human melanoma lines (less than 1%) and the highest levels in some Burkitt's lymphoma lines (up to 97%). In these latter tumours the induction of apoptosis is clearly a major component of the hyperthermic response.

Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors.

Rabbit chondrocyte cultures on plastic dishes are capable of depositing a cartilaginous matrix, although the matrix does not calcify unless high levels of phosphate are added to the medium. In the present study, we cultivated a pelleted mass of rabbit growth-plate chondrocytes in the presence of Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 50 micrograms of ascorbic acid per ml in a plastic centrifuge tube. These cells proliferated for several generations and then reorganized into a cartilage-like tissue that calcified without additional phosphate. The deposition of minerals was observed only after synthesis of a short-chain collagen and alkaline phosphatase. Serum factors were required for the increases in alkaline phosphatase and calcium contents. 5-Bromo-2'-deoxyuridine abolished the increases in uronic acid, alkaline phosphatase, and calcium contents. Transforming growth factor beta, at very low concentrations, suppressed the expression of the mineralization-related phenotype by chondrocytes. These results suggest that cartilage-matrix calcification can be controlled by growth factor(s) and that chondrocytes induce the mineralization of extracellular matrix when terminal differentiation is permitted in the absence of an artificial substrate.

Studies on 125I-alpha bungarotoxin binding sites in rat brain.

The distribution and properties of 125I-alpha bungarotoxin (125I-alpha BTX) binding in rat brain using micropunched tissue homogenates were examined with a binding technique. Highest level of 125I-alpha BTX binding was observed in the hypothalamus, followed by hippocampus, cortex, globus pallidus, nucleus caudatus and nucleus accumbens. Although high levels of 3H-quinuclidinyl benzilate (3H-QNB) binding in the striatum were found at dorso-lateral sites and low levels in the central regions, the level of 125I-alpha BTX binding within the striatum was almost equal. There was no significant correlation between 125I-alpha BTX binding and choline uptake which could be useful as a marker for functional cholinergic nerve terminals. The inhibition of specific 125I-alpha BTX binding to the hippocampal homogenates by nicotine, d-tubocurarine and other nicotine drugs was studied. The results of these studies suggest that alpha BTX binding sites in the brain are not likely the binding sites of nicotine or the physiological ACh R sites.