Workshift Changes in Hydration Status During Wildfire Suppression.

OBJECTIVE: Document wildland firefighters (WLFFs) hydration status during a singular workshift (13.7 ±â€Š1.4 hours). METHODS: WLFF researchers documented real-time WLFF (n = 71) urine metrics and fluid consumption. Body weight and blood samples (n = 25) were also collected. Two-tailed dependent t tests determined statistical significance (P < 0.05). RESULTS: Body weight significantly decreased (-0.3 ±â€Š1.1%, P > 0.05). Fluid consumption totaled 6.2 ±â€Š2.3 L including food and 5.0 ±â€Š2.1 L without food. Morning versus afternoon urine frequency (2.6 ±â€Š1.3, 3.1 ±â€Š1.9 voids), urine volume (1.2 ±â€Š0.7, 1.3 ±â€Š0.8 L), urine volume per void (440 ±â€Š157, 397 ±â€Š142 mL), and urine specific gravity (1.010 ±â€Š0.007, 1.010 ±â€Š0.007) were not significantly different (P > 0.05). Pre- to post-workshift serum chloride (103.2 ±â€Š1.9, 101.4 ±â€Š1.7 mM) and blood glucose (5.2 ±â€Š0.4, 4.5 ±â€Š0.7 mM) significantly decreased (P < 0.05), while serum sodium (141.5 ±â€Š2.4, 140.8 ±â€Š2.0 mM) and serum potassium (4.3 ±â€Š0.3, 4.2 ±â€Š0.3 mM) remained stable (P > 0.05). CONCLUSIONS: WLFFs can ingest fluid and food amounts that maintain workshift euhydration and electrolyte status.

Influence of Fluid Delivery Schedule and Composition on Fluid Balance, Physiologic Strain, and Substrate Use in the Heat.

INTRODUCTION: Wildfire suppression is characterized by high total energy expenditure and water turnover rates. Hydration position stands outline hourly fluid intake rates. However, dose interval remains ambiguous. We aimed to determine the effects of microdosing and bolus-dosing water and microdosing and bolus-dosing carbohydrate-electrolyte solutions on fluid balance, heat stress (physiologic strain index [PSI]), and carbohydrate oxidation during extended thermal exercise. METHODS: In a repeated-measures cross-over design, subjects completed four 120-min treadmill trials (1.3 m·s-1, 5% grade, 33°C, 30% relative humidity) wearing a US Forest Service wildland firefighter uniform and a 15-kg pack. Fluid delivery approximated losses calculated from a pre-experiment familiarization trial, providing 22 doses·h-1 or 1 dose·h-1 (46±11, 1005±245 mL·dose-1). Body weight (pre- and postexercise) and urine volume (pre-, during, and postexercise) were recorded. Heart rate, rectal temperature, skin temperature, and steady-state expired air samples were recorded throughout exercise. Statistical significance (P<0.05) was determined via repeated-measures analysis of variance. RESULTS: Total body weight loss (n=11, -0.6±0.3 kg, P>0.05) and cumulative urine output (n=11, 677±440 mL, P>0.05) were not different across trials. The micro-dosed carbohydrate-electrolyte trial sweat rate was lower than that of the bolus-dosed carbohydrate-electrolyte, bolus-dosed water, and microdosed water trials (n=11, 0.8±0.2, 0.9±0.2, 0.9±0.2, 0.9±0.2 L·h-1, respectively; P<0.05). PSI was lower at 60 than 120 min (n=12, 3.6±0.7 and 4.5±0.9, respectively; P<0.05), with no differences across trials. The carbohydrate-electrolyte trial's carbohydrate oxidation was higher than water trial's (n=12, 1.5±0.3 and 0.8±0.2 g·min-1, respectively; P<0.05), with no dosing style differences. CONCLUSIONS: Equal-volume diverse fluid delivery schedules did not affect fluid balance, PSI, or carbohydrate oxidation during extended thermal work.

Total Energy Intake and Self-Selected Macronutrient Distribution During Wildland Fire Suppression.

INTRODUCTION: Wildland firefighters (WLFF) work long hours in extreme environments, resulting in high daily total energy expenditure. Increasing work-shift eating episodes and/or providing rations that promote convenient eating has shown augmented self-selected work output, as has regular carbohydrate (CHO) consumption. It remains unclear how current WLFF feeding strategies compare to more frequent nutrient delivery. Our aim was to determine WLFFs' self-selected field total energy intake (TEI), composition, and feeding patterns during wildland fire suppression shifts. METHODS: WLFF were deployed to fire incidents across the United States throughout the 2018 fire season. Preshift food inventories collected at basecamp provided item-specific nutrient content (kilocalories, CHO, fat, protein). Work shift consumption (TEI, feeding frequency, episodic composition) was monitored in real time by field researchers on fireline via observational data capture using mobile tablets. Shift work output was also quantified via actigraph accelerometry. RESULTS: Eighty-six WLFF (27.5±6.4 y; 16 female, 70 male) worked 14.0±1.1 h shifts, with a TEI of 6.3±2.5 MJ (1494±592 kcal) (51±10, 37±9, 13±4% for CHO, fat, and protein, respectively). WLFF averaged 4.3±1.6 eating episodes (1.4±1.3 MJ [345±306 kcal] and 44±38 g CHO·episode-1). WLFF who consumed >20 kcal·kg-1 averaged less sedentary activity than those consuming <16 kcal·kg-1. Data are presented as mean±SD. CONCLUSIONS: Not including fire camp meals (breakfast, dinner), the present work-shift TEI approximates 33% of previously determined WLFF total energy expenditure and demonstrates that current WLFF consumption patterns may not deliver adequate nutrients for occupational demands.

Age-dependent increase of blood-brain barrier permeability and neuron-binding autoantibodies in S100B knockout mice.

S100B is a calcium-sensor protein that impacts multiple signal transduction pathways. It is widely considered to be an important biomarker for several neuronal diseases as well as blood-brain barrier (BBB) breakdown. In this report, we demonstrate a BBB deficiency in mice that lack S100B through detection of leaked Immunoglobulin G (IgG) in the brain parenchyma. IgG leaks and IgG-binding to selected neurons were observed in S100B knockout (S100BKO) mice at 6 months of age but not at 3 months. By 9 months, IgG leaks persisted and the density of IgG-bound neurons increased significantly. These results reveal a chronic increase in BBB permeability upon aging in S100BKO mice for the first time. Moreover, coincident with the increase in IgG-bound neurons, autoantibodies targeting brain proteins were detected in the serum via western blots. These events were concurrent with compromise of neurons, increase of activated microglia and lack of astrocytic activation as evidenced by decreased expression of microtubule-associated protein type 2 (MAP2), elevated number of CD68 positive cells and unaltered expression of glial fibrillary acidic protein (GFAP) respectively. Results suggest a key role for S100B in maintaining BBB functional integrity and, further, propose the S100BKO mouse as a valuable model system to explore the link between chronic functional compromise of the BBB, generation of brain-reactive autoantibodies and neuronal dysfunctions.

D2-40 is expressed on the luminal surface of pulmonary airspaces in normal developing and adult lung but is lost in conditions associated with intra-alveolar infiltrates.

The D2-40 antigen is a glycosylated sialomucin that is strongly expressed by lymphatic endothelial cells. Recently we observed the expression of D2-40 on the luminal surface of pulmonary airspaces in lung sections. The aim of the study was to assess the expression of D2-40 antigen in normal lung development and in various pathologic conditions in which abnormal alveolar infiltrates were present. Formalin-fixed lung tissue was selected from 42 fetal/neonatal autopsy cases ranging in gestational age from 12 to 41 weeks and from 10 adult lungs. In the fetal/neonatal group, 22 cases were histologically normal, whereas 20 were abnormal (including cases of pneumonia, alveolar hemorrhage, meconium aspiration, pulmonary hypoplasia, and pulmonary interstitial emphysema). In the adult group, 5 cases were histologically normal and 5 had pneumonia. Immunohistochemical staining was performed on all cases using antibody to D2-40. All cases of normal fetal/neonatal lung and normal adult lung showed diffuse strong expression of D2-40 on the luminal surface of the alveolar lining cells. D2-40 expression was also noted on the bronchiolar lining cells of normal fetal/neonatal lung. In all cases in which there was an abnormal infiltrate or foreign material within the airspaces, expression of D2-40 was lost in the alveolar lining. The production of the D2-40 antigen in the alveolar lining occurs as early as 12 weeks gestation and continues to be present throughout all other stages of lung development, as well as in adult lung. These results suggest that D2-40 may have a cell membrane protective function.

PSAPP mice exhibit regionally selective reductions in gliosis and plaque deposition in response to S100B ablation.

BACKGROUND: Numerous studies have reported that increased expression of S100B, an intracellular Ca2+ receptor protein and secreted neuropeptide, exacerbates Alzheimer's disease (AD) pathology. However, the ability of S100B inhibitors to prevent/reverse AD histopathology remains controversial. This study examines the effect of S100B ablation on in vivo plaque load, gliosis and dystrophic neurons. METHODS: Because S100B-specific inhibitors are not available, genetic ablation was used to inhibit S100B function in the PSAPP AD mouse model. The PSAPP/S100B-/- line was generated by crossing PSAPP double transgenic males with S100B-/- females and maintained as PSAPP/S100B+/- crosses. Congo red staining was used to quantify plaque load, plaque number and plaque size in 6 month old PSAPP and PSAPP/S100B-/- littermates. The microglial marker Iba1 and astrocytic marker glial fibrillary acidic protein (GFAP) were used to quantify gliosis. Dystrophic neurons were detected with the phospho-tau antibody AT8. S100B immunohistochemistry was used to assess the spatial distribution of S100B in the PSAPP line. RESULTS: PSAPP/S100B-/- mice exhibited a regionally selective decrease in cortical but not hippocampal plaque load when compared to PSAPP littermates. This regionally selective reduction in plaque load was accompanied by decreases in plaque number, GFAP-positive astrocytes, Iba1-positive microglia and phospho-tau positive dystrophic neurons. These effects were not attributable to regional variability in the distribution of S100B. Hippocampal and cortical S100B immunoreactivity in PSAPP mice was associated with plaques and co-localized with astrocytes and microglia. CONCLUSIONS: Collectively, these data support S100B inhibition as a novel strategy for reducing cortical plaque load, gliosis and neuronal dysfunction in AD and suggest that both extracellular as well as intracellular S100B contribute to AD histopathology.

Lymphatic invasion identified by monoclonal antibody D2-40, younger age, and ulceration: predictors of sentinel lymph node involvement in primary cutaneous melanoma.

OBJECTIVES: To assess whether lymphatic invasion identified by immunostaining with monoclonal antibody (Mab) D2-40 in primary cutaneous melanomas correlates with other clinicopathologic factors and to assess whether lymphatic invasion is a potential predictor of sentinel lymph node (SLN) status. DESIGN: Retrospective case-series study. SETTING: Academic referral center. Patients Ninety-six consecutive patients with primary cutaneous melanomas 1 mm thick or greater with adequate pathologic material available for immunohistochemical studies and SLN biopsy. MAIN OUTCOME MEASURES: Association between lymphatic invasion identified by immunostaining with Mab D2-40 in primary cutaneous melanoma and correlation with the clinicopathologic features and the association of all of the factors with SLN status. RESULTS: Lymphatic invasion identified by immunostaining with Mab D2-40 was significantly associated with deeper Clark level of invasion (P < .001), and greater Breslow tumor thickness (P = .01) SLN positivity was identified in 23 of 96 cases (24%). At univariate analysis, younger age (P = .03), ulceration (P < .006), lymphatic invasion (P < .02), deeper Clark level of invasion (P < .008), Breslow tumor thickness (P = .008), and tumor site on the trunk (P = .02) were significantly associated with SLN metastases. At multivariate analysis, only younger age (P = .04), ulceration (P = .03), and lymphatic invasion detected by immunostaining with Mab D2-40 (P = .01) were significantly associated with SLN positivity. The probability of SLN positivity was 13% when all 3 independent prognostic factors yielded negative findings and increased to 61% when all 3 variables yielded positive findings. CONCLUSIONS: Breslow tumor thickness, Clark level of invasion, and tumor site on the trunk predicted SLN status at univariate analysis. Multivariate regression analysis showed that lymphatic invasion identified by immunostaining with Mab D2-40, younger age, and ulceration were the only independent prognostic factors. The most significant predictor of SLN metastasis was the positivity of all 3 independent prognostic factors (61%). Findings of this study suggest that assessment of lymphatic invasion by immunostaining with Mab D2-40 with other clinicopathologic factors can be used to identify patients who could be spared the need for SLN biopsy.

Significance of lymph vessel invasion identified by the endothelial lymphatic marker D2-40 in node negative breast cancer.

Monoclonal antibody D2-40, a marker of lymphatic endothelium, identifies tumor emboli in lymph vessels. The aim of the study was to assess whether D2-40+ lymph vessel invasion (LVI) correlates with clinicopathologic factors including lymphovascular invasion (LVI) as assessed by haematoxylin and eosin-stained sections (H&E+ or H&E-) and to assess the prognostic significance in node-negative breast cancer. The study group consisted of 303 node-negative breast cancer patients that had a median follow-up of 7.6 years. Clinical and pathological data were retrieved from the Henrietta Banting database. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections of the primary invasive carcinoma using D2-40. Immunostaining with CD31 was performed on the discordant cases that were H&E+/D2-40-. D2-40+ lymph vessel invasion was detected in 82/303 (27%) cases. The foci of lymphatic invasion occurred predominantly at the invasive front of the tumor. The absence of D2-40 and CD31 in 13/17 discordant cases was suggestive of retraction artefact. D2-40+ lymph vessel invasion correlated significantly with age (P=0.0003), tumor size (P=0.005), histological grade (P=0.0001), H&E+ (P=<0.0001) and estrogen receptor status (P=0.005) but not with histological type or progesterone receptor status. Multivariate analysis revealed that D2-40+ lymph vessel invasion was the only significant predictor of distant recurrence. There was no significant association between D2-40 status and local recurrence (P=0.752) or regional recurrence (P=0.13). Both D2-40+lymph vessel invasion (P=0.009) and H&E+LVI cases (P=0.02) were associated with overall shorter survival in univariate analysis. These data indicate that D2-40 identifies lymphatic invasion in breast tumors and is a significant predictor of outcome in breast cancer.

Biological significance of occult micrometastases in histologically negative axillary lymph nodes in breast cancer patients using the recent American Joint Committee on Cancer breast cancer staging system.

The biological significance of occult metastases in axillary lymph nodes of breast cancer patients is controversial. The purpose of the study was to determine the prognostic significance of occult micrometastases using the current American Joint Committee on Cancer (AJCC) staging system in a cohort of women with node-negative breast cancer, of whom 5% received adjuvant systemic therapy and who all had long-term follow-up. We studied a cohort of 214 consecutive histologically node-negative breast cancer patients with a median follow-up of 8 years. Blocks of the axillary lymph nodes were assessed for occult micrometastases by examination of an additional hematoxylin-eosin-stained slide and by immunohistochemical staining using an antibody to low molecular weight keratin. Occult metastases were classified according to the sixth edition of the AJCC cancer staging manual. We examined the prognostic effects of occult micrometastases and other clinicopathologic features on recurrence outside the breast with disease-free interval (DFI) and survival from breast cancer with disease-specific survival (DSS). Cytokeratin-positive tumor cells were identified in the lymph nodes in 29 of 214 cases (14%). Two cases had isolated tumor cells and no cluster larger than 0.2 mm [pN0(i+)], whereas 27 of 214 (13%) had micrometastases (larger than 0.2 mm and

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours.

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell-cell interactions among pre-meiotic germ cells and immature Sertoli cells.

Prognostic significance of circulating tumour cells enumerated after filtration enrichment in early and metastatic breast cancer patients.

BACKGROUND: We previously found a higher incidence of circulating tumour cells (CTCs) in women with metastatic breast cancer compared to early disease. In this study, we present follow-up data to explore the prognostic significance of these findings. METHODS: CTCs were quantified by immunostaining and direct visualization after centrifugation and filtration enrichment of peripheral blood from 131 patients. Time to progression (TTP) and overall survival (OS) were defined as interval from first blood sampling to first documented disease progression, or death respectively. Lifetime data was analysed using Kaplan-Meier method, log-rank test and Cox proportional hazards model. RESULTS: Follow-up data is available for 123 patients. In early disease, median CTC>or=4 best distinguished patients with shorter TTP (p=0.05, log-rank test). In univariate analysis, tumour size, grade, lymphovascular invasion (LVI) and receptor status significantly related to TTP but none of the covariates related to OS. In multivariate analysis, T stage was the only independent predictor of TTP. In metastatic disease, median CTC>or=13 optimally identified patients with shorter TTP (p=0.01). In univariate analysis, median CTC level >or=13 and prior lines of chemotherapy predicted for TTP while in multivariate analysis, median CTC level >or=13 was the only significant independent prognostic factor (p=0.02). No relationship between CTC level and OS was found in this subgroup. CONCLUSION: Median CTC level determined in the course of treatment predicts for TTP in metastatic breast cancer. In early breast cancer, an association was found between CTC level and TTP although this did not reach statistical significance (p=0.05).

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome.

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-alpha, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.

Enhanced susceptibility of S-100B transgenic mice to neuroinflammation and neuronal dysfunction induced by intracerebroventricular infusion of human beta-amyloid.

S-100B is an astrocyte-derived protein that is increased in focal areas of the brain most severely affected by neuropathological changes in Alzheimer's disease (AD). Cell-based and clinical studies have implicated S-100B in progression of a pathologic, glial-mediated pro-inflammatory state in the CNS. However, the relationship between S-100B levels and susceptibility to AD-relevant neuroinflammation and neuronal dysfunction in vivo has not been determined. To test the hypothesis that overexpression of S-100B increases vulnerability to beta-amyloid (Abeta)-induced damage, we used S-100B-overexpressing transgenic (Tg) and S-100B knockout (KO) mice in a mouse model that involves intracerebroventricular infusion of human oligomeric Abeta1-42. This model mimics many features of AD, including robust neuroinflammation, Abeta plaques, synaptic damage and neuronal loss in the hippocampus. S-100B Tg, KO, and wild-type (WT) mice were infused with Abeta for 28 days, sacrificed at 60 days, and hippocampal endpoints analyzed. We found that Tg mice showed increased vulnerability to Abeta-induced neuropathology relative to either WT or KO mice. Specifically, Tg mice exhibited enhanced glial activation and neuroinflammation, increased nitrotyrosine staining (a marker of glial-induced neuronal damage), and more pronounced loss of synaptic markers. Interestingly, Tg mice showed no significant differences in Abeta plaque burden compared with WT or KO mice, suggesting that, as in the human situation, the severity of neuronal dysfunction did not correlate with amyloid deposition. Our data are consistent with a model in which S-100B overexpression in AD enhances glial activation and leads to an augmented neuroinflammatory process that increases the severity of neuropathologic sequelae.

Detection of lymphatic invasion in primary melanoma with monoclonal antibody D2-40: a new selective immunohistochemical marker of lymphatic endothelium.

OBJECTIVES: To identify the presence of lymphatic invasion in primary cutaneous melanoma using monoclonal antibody D2-40, a marker of lymphatic endothelium, and to correlate the presence of lymphatic invasion with other clinicopathologic characteristics of the tumors. DESIGN: Retrospective melanoma case series study comparing conventional hematoxylin-eosin staining with D2-40 immunostaining for detection of lymphatic invasion. SETTING: Departments of Pathology and Dermatology, Sunnybrook and Women's College Health Sciences Center, University of Toronto, Toronto, Ontario. Patients Forty-four consecutive cases of primary cutaneous melanoma with a tumor thickness greater than 0.75 mm were examined for presence of lymphatic invasion. RESULTS: Seven (16%) of 44 melanomas showed the presence of lymphatic invasion under immunostaining with D2-40. In 2 cases, subepidermal lymphatic involvement was present; in 5 cases lymphatic invasion was noted within the tumor, including 1 case of additional lymphatic invasion at the invasive edge of the tumor. Lymphatic invasion was not detected on routine hematoxylin-eosin staining. We observed a trend in the association between lymphatic invasion and 2 markers of tumor aggressiveness, namely, a deeper Clark level and increased frequency of ulceration, which suggests that lymphatic invasion detected with D2-40 may indicate a poor prognosis. CONCLUSIONS: Immunostaining with D2-40 increases the frequency of detection of lymphatic invasion relative to conventional hematoxylin-eosin staining in primary melanoma. Future outcome data will determine the prognostic significance of lymphatic invasion detected by D2-40 immunostaining.

S100B expression modulates left ventricular remodeling after myocardial infarction in mice.

BACKGROUND: S100B, a 20-kDa, Ca2+-binding dimer, is a putative intrinsic negative regulator of myocardial hypertrophy expressed after myocardial infarction. S100B-overexpressing transgenic (TG) and S100B-knockout (KO) mice have been generated to assess the consequences of S100B expression and altered hypertrophy after infarction. METHODS AND RESULTS: We compared 21 wild-type (WT), 20 TG, and 24 KO mice over 35 days after experimental myocardial infarction with sham-operated controls (n=56). Of those, 4 WT-infarcted mice, 7 TG-infarcted mice, and 1 KO-infarcted mouse and no sham-operated mice died during the observation period. Among survivors, echocardiography, hemodynamic studies, and postmortem examination indicated that the WT and KO groups of infarcted mice mounted a hypertrophic response that was augmented in KO mice. The S100B-overexpressing TG group did not develop hypertrophy but demonstrated increased apoptosis. The postinfarct end-diastolic pressure was lower in KO mice than in WT mice, in accordance with other structural, hemodynamic, and functional parameters, which suggests that abrogation of S100B expression augmented hypertrophy, decreased apoptosis, and was beneficial to preservation of cardiac function within this time frame. CONCLUSIONS: S100B regulates the hypertrophic response and remodeling in the early postinfarct period and represents a potential novel therapeutic target.

Chronic gliosis induced by loss of S-100B: knockout mice have enhanced GFAP-immunoreactivity but blunted response to a serotonin challenge.

Serotonin (5-HT) can induce a release of intraglial S-100B and produce a change in glial morphology. Because S-100B can inhibit polymerization of glial fibrillary acidic protein (GFAP), we hypothesize that glial reactivity may reflect the loss of intraglial S-100B. Adult male transgenic S-100B homozygous knockout (-/-) mice (KO) and wild-type CD-1 (WT) mice were studied. S-100B-immunoreactivity (IR) was seen in the brain tissue of WT (CD-1) but not S-100B KO (-/-) mice. GFAP-IR was seen in both WT (CD-1) and S-100B KO (-/-) glia cells, but S-100B KO (-/-) GFAP-IR cells appeared larger, darker, and more branched than in WT (CD-1). To compare the response of GFAP-IR cells to 5-HT in S-100B KO (-/-) and WT (CD-1) mice, we injected animals with para-chloroamphetamine (PCA) over 2 days (5 and 10 mg/ml). PCA is a potent 5-HT releaser which can induce gliosis in the rodent brain. In WT (CD-1) mice, the size, branching, and density of GFAP-IR cells were significantly increased after PCA injections. No increase in GFAP-IR activation was seen in the S-100B KO (-/-) after PCA injections. Cell-specific densitometry (set at a threshold of 0-150 based on a scale of 255) in these animals statistically showed an increase in GFAP-IR after PCA injections in WT (CD-1) but not S-100B KO (-/-) mice. These results are consistent with the hypothesis that 5-HT may modulate glial morphology by inducing a release of intracellular S-100B, and this pathway is inoperable in the S-100B KO (-/-).

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis.

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T1-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T1-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T2-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.

Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Enumeration of circulating tumor cells in the blood of breast cancer patients after filtration enrichment: correlation with disease stage.

The biological and clinical significance of circulating tumor cells (CTC) in the peripheral blood of breast cancer patients is not known. To study this question, we used a direct visualization assay to correlate the number of CTC with disease stage and progression. The CTC were enriched from the nucleated cell fraction by filtration and enumerated visually following immunostaining with anti-cytokeratin 8 (CK8) antibody CAM 5.2. In mixing experiments, we achieved a limit of detection of 5 MCF7 cells per 5 ml of blood or 5 x 10(7) peripheral blood leukocytes (PBL). We did not detect CTC in any control subjects (0/20). In 131 breast cancer patients, we found a higher incidence of CTC in patients with distant metastatic 36/51 (71%) than those with node-positive 17/36 (47%) (p = 0.026), or node-negative 17/44 (39%) (p = 0.001) disease. The distribution of the highest numbers of CTC observed in individual patients by repeated sampling over time ranged from 1 to 700 per 5 ml of blood with a trend toward higher numbers in those with distant metastases. In comparison with previous studies of equal specificity, based on a similar absence of CTC in controls, we report a higher incidence of CTC in node-negative and node-positive patients, suggesting a more frequent detection of CTC by our approach. This higher incidence was achieved, in part, by repeated sampling of our study population over time. Our results support the concept that CTC can be detected and enumerated in peripheral blood and that this minimally invasive assay merits further evaluation as a potential prognostic indicator and marker of disease progression.

Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation.

The S100B protein belongs to the S100 family of EF-hand calcium binding proteins implicated in cell growth and differentiation. Here, we show that in the developing and the adult mouse brain, S100B is expressed in oligodendroglial progenitor cells (OPC) committed to differentiate into the oligodendrocyte (OL) lineage. Nuclear S100B accumulation in OPC correlates with the transition from the fast dividing multipotent stage to the morphological differentiated, slow proliferating, pro-OL differentiation stage. In the adult, S100B expression is down-regulated in mature OLs that have established contacts with their axonal targets, suggesting a nuclear S100B function during oligodendroglial cells maturation. In vitro, the morphological transformation and maturation of pro-OL cells are delayed in the absence of S100B. Moreover, mice lacking S100B show an apparent delay in OPC maturation in response to demyelinating insult. We propose that nuclear S100B participates in the regulation of oligodendroglial cell maturation.