Alternation between short- and long photoperiod reveals hypothalamic gene regulation linked to seasonal body weight changes in Djungarian hamsters (Phodopus sungorus).

Djungarian hamsters are able to reduce their body weight by more than 30% in anticipation of the winter season. This particular adaptation to extreme environmental conditions is primarily driven by a natural reduction in day length and conserved under laboratory conditions. We used this animal model to investigate hypothalamic gene expression linked to body weight regulation behind this physiological phenomenon. After an initial collective short photoperiod (SP) adaptation for 14 weeks from a preceding long photoperiod (LP), hamsters were re-exposed to LP for either 6 or 14 weeks, followed by a second re-exposure to SP for 8 weeks. Our data showed that re-exposure to LP led to an increase in body weight. In the hypothalamus Dio2, Vimentin, Crbp1 and Grp50 expression increased, whereas expression of Dio3, Mct8 and Srif decreased. The changes in body weight and gene expression were reversible in most hamsters after a further re-exposure to SP following 6 or 14 weeks in LP. Interestingly, after 14 weeks in LP, body weight loss was pronounced in six hamsters re-exposed to SP, but five hamsters did not respond. In nonresponding hamsters, a different gene expression pattern was manifested, with the exception of Dio2, which was reduced not only in SP re-exposed hamsters, but also in hamsters maintained in LP. Taken together, these data suggest that body weight regulation appears to be tightly linked to a co-ordinated regulation of several genes in the hypothalamus, including those involved in thyroid hormone metabolism.

Dataset of liver proteins changed in eu- and hypothyroid female rats upon in vivo exposure to hexabromocyclododecane (HBCD).

Female Wistar rats with different thyroid status (eu-, hypothyroid) were exposed to 0, 3 or 30 mg/kg body weight of the flame retardant HBCD for 7 days. Changes in protein patterns obtained by 2D-DIGE were evaluated, and different animal groups compared taking into account their exposure and thyroid status. Proteins significantly altered in abundance in any of these comparisons were identified by mass spectrometry. These data, together with hormone data of the animals, are discussed in "Hexa-bromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats" (Miller et al., 2016) [1].

Hexabromocyclododecane (HBCD) induced changes in the liver proteome of eu- and hypothyroid female rats.

Hexabromocyclododecane (HBCD) is a brominated flame retardant known for its low acute toxicity as observed in animal experiments. However, HBCD exposure can affect liver functioning and thyroid hormone (TH) status. As exact mechanisms are unknown and only limited toxicological data exists, a gel-based proteomic approach was undertaken. In a eu- and hypothyroid female rat model, rats were exposed to 3 and 30 mg/kg bw/day HBCD for 7 days via their diet, and exposure was related to a range of canonical endpoints (hormone status, body weight) available for these animals. Alterations in the liver proteome under HBCD exposure were determined in comparison with patterns of control animals, for both thyroid states. This revealed significantly changed abundance of proteins involved in metabolic processes (gluconeogenesis/glycolysis, amino acid metabolism, lipid metabolism), but also in oxidative stress responses, in both euthyroid and hypothyroid rats. The results provide a more detailed picture on the mechanisms involved in these alterations, e.g. at the protein level changes of the proposed influence of HBCD on the lipid metabolism. Present results show that proteomic approaches can provide further mechanistic insights in toxicological studies.

Gender specific differences in the liver proteome of rats exposed to short term and low-concentration hexabromocyclododecane (HBCD).

The influence of short term (7-day) exposure of male rats to the brominated flame retardant hexabromocyclododecane (HBCD) was studied by investigation of the liver proteome, both in euthyroid and hypothyroid rats and by comparing results with general data on animal physiology and thyroid hormone, leptin, insulin and gonadotropin concentrations determined in parallel. Proteome analysis of liver tissue by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed that only small protein pattern changes were induced by exposure in males, on just a few proteins with different functions and not involved in pathways in common. This is in contrast to previous findings in similarly exposed eu- and hypothyroid female rats, where general metabolic pathways had been shown to be affected. The largest gender-dependent effects concerned basal concentrations of liver proteins already in control and hypothyroid animals, involving mainly the pathways which were also differently affected by HBCD exposure. Among them were differences in lipid metabolism, which - upon exposure to HBCD - may also be the reason for the considerably higher ratio of γ-HBCD accumulated in white adipose tissue of exposed female rats compared to males. The results further elucidate the already suggested different sensitivity of genders towards HBCD exposure on the protein level, and confirm the need for undertaking toxicological animal experiments in both genders.

Sinonasal manifestations of cystic fibrosis: a correlation between genotype and phenotype?

BACKGROUND: Patients with Cystic Fibrosis are prone to develop sinonasal disease. Studies in genotype-phenotype correlations for sinonasal disease are scarce and inconclusive. METHODS: In this observational study several aspects of sinonasal disease were investigated in 104 adult patients with CF. In each patient a disease specific quality of life questionnaire (RSOM-31), nasal endoscopy and a CT scan of the paranasal sinuses were performed. Patients were divided into two groups, class I-III mutations and class IV-V mutations, based on their CFTR mutations. RESULTS: The prevalence of rhinosinusitis in adult patients with CF was 63% and the prevalence of nasal polyps 25%. Patients with class I-III mutations had significantly smaller frontal sinuses, sphenoid sinuses, more opacification in the sinonasal area and more often osteitis/neoosteogenesis of the maxillary sinus wall compared to patients with class IV and V mutations. CONCLUSION: These data suggest more severe sinonasal disease in patients with class I-III mutations compared to patients with class IV-V mutations.

Importance of bacteriology in upper airways of patients with Cystic Fibrosis.

BACKGROUND: Recently the influence of the upper airways (UAW) on the general health of a patient with Cystic Fibrosis (CF) has been acknowledged. Surprisingly the microbiology of the upper compartment of the airways receives barely any attention in the treatment of CF. The aim of the present study was to investigate the microbiology of the upper airways in adult patients with CF, to correlate these findings with cultures from the lower airways (LAW) and with clinical characteristics. METHODS: In this cross-sectional study bacteriological and clinical data were gathered from 104 adult patients with CF. UAW samples for culture were collected by nasal lavage and middle meatal swabs; LAW cultures were performed on expectorated sputum or cough swabs. Each patient performed the Rhinosinusitis Outcome Measure (RSOM-31). RESULTS: In 72 patients (69.2%) UAW cultures yielded microorganisms other than normal nasal flora and in 50 patients (48.1%) Pseudomonas aeruginosa grew from the UAW cultures. Similarity between UAW and LAW cultures was determined in 50.0% of these 72 patients. In 3 patients P. aeruginosa was cultured from the UAW after successful eradication of P. aeruginosa from the LAW. P. aeruginosa in the UAW did not influence symptoms of sinonasal disease compared to other microorganisms. CONCLUSIONS: Comparison of UAW and LAW cultures in adult patients with CF showed one or more concordant microorganism in 50.0% of the patients. P. aeruginosa was most frequently cultured from the UAW. P. aeruginosa can be cultured from the UAW after eradication therapy which may suggest persistence of P. aeruginosa in the UAW. We feel this is may be a motive to include the UAW in eradication therapy in Cystic Fibrosis.

Perinatal and chronic hypothyroidism impair behavioural development in male and female rats.

A lack of thyroid hormone, i.e. hypothyroidism, during early development results in multiple morphological and functional alterations in the developing brain. In the present study, behavioural effects of perinatal and chronic hypothyroidism were assessed during development in both male and female offspring of hypothyroid rats. To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate; dams starting 2 weeks prior to mating and pups either until the day of killing (chronic hypothyroidism) or only until weaning (perinatal hypothyroidism) to test for reversibility of the effects observed. Neuromotor competence, locomotor activity and cognitive function were monitored in the offspring until postnatal day 71 and were compared with age-matched control rats. Early neuromotor competence, as assessed in the grip test and balance beam test, was impaired by both chronic and perinatal hypothyroidism. The open field test, assessing locomotor activity, revealed hyperactive locomotor behavioural patterns in chronic hypothyroid animals only. The Morris water maze test, used to assess cognitive performance, showed that chronic hypothyroidism affected spatial memory in a negative manner. In contrast, perinatal hypothyroidism was found to impair spatial memory in female rats only. In general, the effects of chronic hypothyroidism on development were more pronounced than the effects of perinatal hypothyroidism, suggesting the early effects of hypothyroidism on functional alterations of the developing brain to be partly reversible and to depend on developmental timing of the deficiency.

Inflammatory cells seem not to be involved in idiopathic rhinitis.

Mucosal inflammatory cellular infiltrates are correlated with nasal complaints in symptomatic allergic rhinitis. Some authors suggest inflammation of a neurogenic or immunogenic nature as an underlying disorder for idiopathic rhinitis (IR). We looked at the possible involvement of inflammatory cells in the pathogenesis of IR. Nasal biopsies were taken from sixty-five IR patients with significant nasal complaints and from twenty healthy controls with no nasal complaints. Inflammatory cells were quantified using monoclonal antibodies directed against lymphocytes, antigen-presenting cells, eosinophils, macrophages, monocytes, mast cells and other IgE-positive cells. No significant differences were found, for any cell, between IR patients and controls. We conclude that inflammatory cells do not seem to play an important role in this meticulously characterised group of IR patients.

Pollutional and meteorological factors are closely related to complaints of non-allergic, non-infectious perennial rhinitis patients: a time series model.

BACKGROUND: Epidemiological studies in the past have focused on meteorological conditions, pollution and pollen and their relationship with symptoms of bronchial hyper-reactivity, however, there are no epidemiological studies which examine a wide range of such factors and determine their role in nasal hyper-reactivity. OBJECTIVE: To investigate whether environmental factors can influence symptomatology in non-allergic non-infectious perennial rhinitis (NANIPER) patients, who suffer primarily from nasal hyper-reactivity symptoms. METHODS: We studied 16 non-smoking NANIPER patients and seven non-smoking controls during a 218-day study period (March-October) by means of daily symptom scores and visual analogue scales for the subsets patency, secretions and sneezing, and compared them to seven primary factors which affected 'symptoms' and 10 secondary factors which affected primary factors only. RESULTS: The mean symptom scores in the NANIPER and control groups were 2.17 and 0.13, respectively. In NANIPER, the highest correlations of primary factors with symptomatology were found for symptom scores and sneezing with minimum daytime temperature (r = -0.62 and -0.45, respectively), ozone and NO concentrations. Patency and secretions were associated with minimum daytime temperature (r = -0.39 and 0.32, respectively). Time series analysis, however, correcting for several confounders such as autocorrelated symptomatology, showed that minimum daytime temperature and daytime relative humidity made an independent contribution to symptoms. In the control group, correlations were much lower, though present. Time series analysis was not possible. CONCLUSIONS: We conclude that in a mild climate with relatively low levels of pollution, minor pollution and meteorological disturbances result in substantial changes in nasal reactivity symptoms in NANIPER patients, but not controls, irrespective of other factors such as allergy or infection.

Forced expiration through the nose is a stimulus for NANIPER but not for controls.

OBJECTIVE: Is forced expiration through the nose a mechanical stimulus to which patients with nasal hyperreactivity react? Do parameters, such as peak nasal expiratory flow rate (PNEF), influence nasal airway resistance (NAR) in these patients? METHOD: NAR, mucus production and sneezing were measured on 2 occasions two weeks apart. Measurements were conducted before and during a period of 10 minutes after 3 repeated PNEFs in 15 non-allergic non-infectious perennial rhinitis (NANIPER) patients suffering from nasal hyperreactivity, and in 15 controls. RESULTS: In NANIPER versus controls PNEF measurements attributed to a statistically significant increase in NAR. The main effect was within the first minute after stimulus, suggesting a neuronal mechanism. Mucus secretions and sneezing were hardly present. PNEF (highest of 3) and bronchial peak expiratory flow rate (BpEFR) are lower in NANIPER than controls but are correlated. Impaired bronchial capacity is likely to influence PNEF, resulting in a lower decrease of nasal patency. CONCLUSION: PNEF depends on BpEFR and is an adequate mechanical stimulus for NANIPER patients, but not for non-rhinitic controls, resulting in a brief increase in NAR.

Long-term effects of corticosteroid nasal spray on nasal inflammatory cells in patients with perennial allergic rhinitis.

BACKGROUND: The effect of long-term topical nasal corticosteroid therapy on nasal inflammatory cells is unclear. OBJECTIVES: To investigate the long-term effect of fluticasone propionate aqueous nasal spray (FPANS) on nasal mucosal inflammatory cells and efficacy in a 1-year study in patients with perennial allergic rhinitis. METHODS: In a 1-year, double-blind, placebo-controlled study of duration we investigated the influence of a topical corticosteroid (FPANS), on Langerhans' cells (CD1a+ cells), T cells, mast cells, eosinophils and macrophages in nasal mucosa in 42 patients with perennial allergic rhinitis. Efficacy was evaluated by nasal symptom score. RESULTS: The FPANS group experienced significantly less sneezing and nasal itching compared with the placebo group. The total symptom score in the FPANS group declined significantly in comparison with baseline (P = 0.007) and placebo group (P = 0.009). After 1 year of active treatment, a significant decrease was seen in the epithelium in numbers of Langerhans' cells, CD3+, CD4+, CD8+ cells, mast cells and eosinophils. In the lamina propria, there was a significant decrease in eosinophils. CONCLUSION: These findings show that FPANS treatment results in a decrease of nasal inflammatory cells. Furthermore, the efficacy of FPANS improves after prolonged treatment.

Intranasal cold dry air is superior to histamine challenge in determining the presence and degree of nasal hyperreactivity in nonallergic noninfectious perennial rhinitis.

The objective of the study was to compare cold dry air (CDA) and histamine in differentiating patients with nonallergic noninfectious perennial rhinitis (NANIPER) from control subjects. Nasal reactivity (nasal patency, mucus production, and sneezing) in 16 symptomatic nonsmoking patients with NANIPER and seven nonsmoking control subjects was measured with standardized CDA and histamine provocation series in a randomized crossover study. Intranasal CDA resulted in increased mucus production and nasal blockage in a dose-dependent manner in patients with NANIPER but not in control subjects. Sneezing did not occur. The reproducibility of CDA for patency and mucus production was good. Sensitivity for CDA was 87% compared with 100% for histamine. However, specificity was 71% for CDA and 0% for histamine. It is concluded that the new standardized intranasal CDA provocation method uses a recognizable natural nonspecific stimulus and seems to be more suitable than histamine for characterizing and assessing the presence and degree of nasal reactivity in NANIPER.

A 1-year placebo-controlled study of intranasal fluticasone propionate aqueous nasal spray in patients with perennial allergic rhinitis: a safety and biopsy study.

In a 1-year, placebo-controlled, double-blind, randomized study the long-term effect of Fluticasone Propionate Aqueous Nasal Spray (FPANS) in 42 patients with a perennial allergic rhinitis was studied with regard to safety and efficacy. Twenty-nine patients completed the entire treatment period. After 1 year of treatment no deleterious changes consequent on therapy were observed in nasal mucosal biopsies. The appearance of the epithelial layer, the degree of cellular infiltration, the extent to which the sinusoids were dilated and the degree of tissue oedema improved or remained unchanged in 93% of the patients of the FPANS group, versus 75% of the placebo group, and worsened in 7% of the FPANS group versus 25% of the placebo group. Assessment of the changes in haematological, biochemical, urinary, plasma cortisol levels, and in the findings during nasal examination revealed no significant differences between the two treatment groups. After 1 year of treatment symptom scores for sneezing, nasal itching, and total symptom score were significantly better in the FPANS treated group (P < 0.05, P < 0.05, P < 0.01). An initial reduction in total symptom score was found after 4 weeks FPANS treatment with a further reduction after 8 months of FPANS treatment. These findings suggest that the maximum efficacy of topical intranasal steroids is reached after long-term treatment, and thus advocates longer usage before treatment is stopped because of presumed inefficacy.

The effect of nasal steroid aqueous spray on nasal complaint scores and cellular infiltrates in the nasal mucosa of patients with nonallergic, noninfectious perennial rhinitis.

Topical corticosteroids are the therapy of choice for nonallergic, noninfectious perennial rhinitis (NANIPER). However, the efficacy of steroid therapy in NANIPER is controversial, as is its mode of action. To our surprise, of 300 patients initially diagnosed as having NANIPER, only 65 reached threshold nasal symptom scores. Patients were randomized into four different treatment regimens: placebo administered twice daily (BD) for 8 weeks, fluticasone propionate aqueous nasal spray (FPANS) (200 microg) once daily (OD) and placebo OD for 8 weeks, FPANS (200 microg) OD and placebo OD for 4 weeks followed by FPANS (200 microg) BD for 4 weeks, and FPANS (200 microg) BD for 8 weeks. A small decrease in nasal symptoms was found, which only reached significance for sneezing in the FPANS 200 microg BD group. A significant dose-dependent decrease in immunocompetent cells was found in nasal biopsy specimens obtained before, after 4 weeks, and after 8 weeks of treatment. We conclude that FPANS did not significantly reduce nasal symptoms in this group of selected NANIPER patients, even though a significant effect on cells in the nasal mucosa was seen.

Dynamics of nasal eosinophils in response to a nonnatural allergen challenge in patients with allergic rhinitis and control subjects: a biopsy and brush study.

BACKGROUND: Eosinophils are thought to play an important role in the symptomatology and pathophysiology of allergic rhinitis. Most quantitative studies on eosinophils in nasal mucosa have focused on the dynamics of eosinophils in the acute and late phases of the allergic reaction by using different cell sampling techniques. Little is known about the dynamics of eosinophils during a more prolonged period of allergen exposure and the activation of eosinophils induced by allergen challenge. OBJECTIVE: The aim of this study was to investigate the dynamics and activation of the eosinophils in the nasal mucosa of patients with an isolated grass pollen allergy during an out-of-season 2-week allergen exposure, mimicking the natural grass pollen season. METHODS: Seventeen patients with isolated grass pollen allergy and four control subjects were challenged daily with the allergen during a 2-week period in the winter. Nasal brush specimens were obtained before provocation and each day during the provocation period. Biopsy specimens were obtained once before, six times during, and once after the provocation period. Preparations made of nasal brush and nasal biopsy specimens were stained with the monoclonal antibody BMK 13 and Giemsa stain as paneosinophil markers and with the monoclonal antibody EG2 to identify activated eosinophils. RESULTS: We found significant increases in the total number of eosinophils and the number of activated eosinophils in the epithelium and lamina propria. These increases were most explicit in the second week. BMK 13 was found to be a paneosinophil marker superior to Giemsa staining. CONCLUSION: Eosinophils are not only involved in the acute and late phases of the allergic reaction but are probably even more involved in the chronic phase.

The lack of effect of benzalkonium chloride on the cilia of the nasal mucosa in patients with perennial allergic rhinitis: a combined functional, light, scanning and transmission electron microscopy study.

BACKGROUND: Controversy still exists about the effect of 0.02% benzalkonium chloride (BKC), a preservative in many nasal sprays, on human nasal epithelium in vivo. OBJECTIVE: To determine the safety of BKC by assessing its effect on the function and morphology of cilia of human nasal epithelium. METHODS: A single-centre, double-blind nasal biopsy study in 22 patients with perennial allergic rhinitis, receiving fluticasone propionate aqueous nasal spray (FPANS) containing BKC, BKC plus placebo or placebo alone for 6 weeks. Before, at two weekly intervals during treatment and 2 weeks after treatment ceased an indigocarmine saccharine transport time (ICST) was performed. RESULTS: ICST results did not significantly vary between the groups. There was no statistical relationship between the number of ciliated cells present and the treatment the patients received. Scanning and transmission electron microscopy examination showed no effects of BKC. CONCLUSION: Despite reports of its ciliostatic effects in vitro, BKC did not have such an effect when it was applied for 6 weeks (with/without fluticasone propionate) to the nasal mucosa of perennial allergic rhinitis patients in vivo.

Effect of 3 months' nasal steroid therapy on nasal T cells and Langerhans cells in patients suffering from allergic rhinitis.

The effect of nasal corticosteroid therapy on allergic rhinitis is uncertain. In a double-blind, placebo-controlled study over 3 months, we investigated the influence of a new corticosteroid spray, fluticasone propionate aqueous nasal spray (FPANS), on Langerhans cells (CD1a+ cells), HLA-DR+ cells, and T cells in nasal mucosa. Efficacy was evaluated by nasal symptom score. This treatment significantly decreased the number of CD1a+ cells and HLA-DR+ cells in the nasal mucosa. Furthermore, a clear trend of decreasing numbers of T cells in nasal epithelium was found. No change in nasal symptom score was found after the treatment period. These findings suggest that fluticasone propionate aqueous nasal spray decreases the antigen presentation in nasal allergy.

The effect of fluticasone propionate aqueous nasal spray on nasal mucosal inflammation in perennial allergic rhinitis.

Mast cell degranulation, and the subsequent recruitment of infiltrating inflammatory cells, such as eosinophils, into the nasal mucosa has long been considered the most important model to explain allergic rhinitis. Several studies show a decrease in the number of eosinophils and possibly also mast cells during local corticosteroid treatment. Over the last decade, a new model to explain allergic inflammation has evolved. In this model, Langerhans' cells and T-cells play an important role. Langerhans' cells possess a high affinity receptor for IgE. In patients with allergic rhinitis, allergen provocation results in stimulation of T-cells by the IgE-positive Langerhans' cells. The T-cells produce a number of cytokines which stimulate IgE production as well as the inflammatory reaction. The number of T-cells is not usually influenced by corticosteroid treatment; however, the function of the T-cells, shown by the spectrum of cytokines produced, is clearly influenced. The cells that are most dramatically affected by local corticosteroid treatment are the Langerhans' cells, which completely disappear during treatment. This decrease suggests that there is a reduction in antigen presentation. The subsequent decrease in T-cell stimulation may result in a reduction of the reactions that are dependent on T-cell-derived mediators.

Mast cells, eosinophils and IgE-positive cells in the nasal mucosa of patients with vasomotor rhinitis. An immunohistochemical study.

Vasomotor rhinitis (VMR) is a disorder of unknown pathogenesis. Forty patients with VMR were carefully selected on the basis of inclusion and exclusion criteria proposed by Mygind and Weeke. Nasal biopsy specimens were taken in the patient group as well as in a group of ten controls. Brush cytology was also taken in the VMR group. Inflammatory cells were identified and counted in the nasal mucosa, with the use of immunohistochemical techniques and a panel of monoclonal antibodies. Eosinophils were studied with the use of BMK13, EG2, and Giemsa. Mast cells were studied with anti-chymase (B7), anti-tryptase (G3) and toluidine blue. Sections were stained with IgE as well. There was no significant difference in the number of eosinophils, mast cells and IgE-positive cells between the two groups. Additionally, in contrast with other reports, in sections that were double-stained with anti-chymase and anti-tryptase, single chymase-positive cells were found.

Dynamics of mast cells in the nasal mucosa of patients with allergic rhinitis and non-allergic controls: a biopsy study.

Mast cell degranulation is thought to be an important component of the pathogenesis of allergic rhinitis. Quantitative studies on mast cells in nasal mucosa after allergen exposure have given widely divergent results, ranging from an overall decrease via redistribution to an overall increase. We investigated this problem by employing a combination of anti-IgE and toluidine blue staining of biopsy specimens. In allergic patients anti-IgE was found to identify all mast cells and toluidine blue to detect mast cells that were not (totally) degranulated. The study was composed of two parts done in different patient groups. In the first part of the study biopsies were performed in 23 patients with isolated grass-pollen allergy, once during natural provocation in the summer and once in the winter. Biopsies were also performed in 12 controls. Non-allergic controls were found to have the same number of mast cells in the lamina propria as asymptomatic allergic patients. The controls seldom have mast cells in the epithelium. The patients with isolated grass-pollen allergy showed an increase in the numbers of mast cells in the lamina propria during natural provocation and the same seemed to occur in the epithelium as well. During natural provocation almost all of the mast cells in the epithelium and half of those in the lamina propria were degranulated. In the second part of the study 17 patients with isolated grass-pollen allergy and four controls were challenged daily with allergen extract during a 2-week period in the winter. During this period biopsies were performed at eight different occasions, i.e. once before, six occasions during and once after the provocation period. The results of this part of the study showed that during provocation mast cells migrate to the surface of the nasal mucosa, where they become degranulated, and that the pool of mast cells in the lamina propria was apparently replenished by migration of mast cells from the vessels in the lamina propria. The total number of mast cells in the lamina propria remained approximately the same while the mast cells residing in an increasingly thick layer measured from the basal membrane into the lamina propria became degranulated. After 2 weeks, 82% of the mast cells in the lamina propria was degranulated and it was only in the deepest layers that some toluidine blue positive cells were found.(ABSTRACT TRUNCATED AT 400 WORDS)