Defining the Histopathological Term Atypical Intraepidermal Melanocytic Proliferation: A Retrospective Cross-Sectional Study.

BACKGROUND: Atypical intraepidermal melanocytic proliferation (AIMP) is a general term assigned to melanocytic proliferations of uncertain biological potential when a definitive histopathological diagnosis cannot be achieved. There are few data available describing the possibility of malignancy of AIMP, or ways to further define diagnosis. OBJECTIVE: To determine the rate of diagnostic change of AIMP to melanoma or melanoma in situ (MIS) after conventional excision. In addition, to determine the role of immunohistochemistry (IHC) in defining AIMP biopsies. METHODS: Retrospective cross-sectional, single-center review of biopsies with a diagnosis of AIMP with a follow-up conventional excision from 2012-2016 was performed. In a separate analysis, a search was performed for AIMP biopsied lesions in which IHC was subsequently performed. RESULTS: The rate of diagnostic change of AIMP to MIS was 4.8% (8/167) after excision. Punch biopsy was a risk factor for diagnostic change to MIS (odds ratio 12.94, confidence interval 2.56-65.38, P = 0.008). The rate of diagnostic change of AIMP biopsies after examining with IHC was 21.3% (34/160) to MIS and 4.4% (7/160) to melanoma. CONCLUSION: The possibility of malignancy of AIMP lesions must be taken into consideration when counseling patients and when planning treatment options. IHC is a useful tool and should be used in the evaluation of AIMP specimens.

Ancient mitochondrial DNA reveals convergent evolution of giant short-faced bears (Tremarctinae) in North and South America.

The Tremarctinae are a subfamily of bears endemic to the New World, including two of the largest terrestrial mammalian carnivores that have ever lived: the giant, short-faced bears Arctodus simus from North America and Arctotherium angustidens from South America (greater than or equal to 1000 kg). Arctotherium angustidens became extinct during the Early Pleistocene, whereas Arctodus simus went extinct at the very end of the Pleistocene. The only living tremarctine is the spectacled bear (Tremarctos ornatus), a largely herbivorous bear that is today only found in South America. The relationships among the spectacled bears (Tremarctos), South American short-faced bears (Arctotherium) and North American short-faced bears (Arctodus) remain uncertain. In this study, we sequenced a mitochondrial genome from an Arctotherium femur preserved in a Chilean cave. Our molecular phylogenetic analyses revealed that the South American short-faced bears were more closely related to the extant South American spectacled bear than to the North American short-faced bears. This result suggests striking convergent evolution of giant forms in the two groups of short-faced bears (Arctodus and Arctotherium), potentially as an adaptation to dominate competition for megafaunal carcasses.

The number of circulating monocytes as biomarkers of the clinical response to methotrexate in untreated patients with rheumatoid arthritis.

BACKGROUND: The aim of this work was to analyze the number and distribution of circulating monocytes, and of their CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in treatment-naive patients with rheumatoid arthritis (RA), and to determine their value in predicting the clinical response to methotrexate (MTX) treatment. METHODS: This prospective work investigated the number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in 52 untreated patients with RA before MTX treatment, and at 3 and 6 months into treatment, using flow cytometry. RESULTS: The absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, were significantly higher in MTX non-responders than in responders and healthy controls before starting and throughout treatment. Responders showed normal numbers of monocytes, and of their subset cells, over the study period. The pre-treatment absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, were found to be predictive of the clinical response to MTX, with a sensitivity and specificity of >70% and >88%, respectively. CONCLUSIONS: Treatment-naive patients with RA showed an anomalous distribution of circulating monocyte subsets, and an anomalous number of cells in each subset. A higher pre-treatment number of circulating monocytes, and higher numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, predict a reduced clinical response to MTX in untreated patients with RA.

Mitochondrial genomes reveal the extinct Hippidion as an outgroup to all living equids.

Hippidions were equids with very distinctive anatomical features. They lived in South America 2.5 million years ago (Ma) until their extinction approximately 10 000 years ago. The evolutionary origin of the three known Hippidion morphospecies is still disputed. Based on palaeontological data, Hippidion could have diverged from the lineage leading to modern equids before 10 Ma. In contrast, a much later divergence date, with Hippidion nesting within modern equids, was indicated by partial ancient mitochondrial DNA sequences. Here, we characterized eight Hippidion complete mitochondrial genomes at 3.4-386.3-fold coverage using target-enrichment capture and next-generation sequencing. Our dataset reveals that the two morphospecies sequenced (H. saldiasi and H. principale) formed a monophyletic clade, basal to extant and extinct Equus lineages. This contrasts with previous genetic analyses and supports Hippidion as a distinct genus, in agreement with palaeontological models. We date the Hippidion split from Equus at 5.6-6.5 Ma, suggesting an early divergence in North America prior to the colonization of South America, after the formation of the Panamanian Isthmus 3.5 Ma and the Great American Biotic Interchange.

Dermatologists in the Emergency Department: A 6-Year Retrospective Analysis.

OBJECTIVE: There are few studies documenting dermatological consultations in the emergency setting. The aim of this study was to evaluate the nature, purpose, and diagnostic accuracy of emergency care physicians in all the dermatology consults evaluated by the Department of Dermatology of the University of Puerto Rico School of Medicine. METHODS: A retrospective analysis of all the consultation reports pertaining to patients evaluated at 4 emergency departments served from July 1, 2007, to June 30, 2013. The data collected from each consultation report consisted of the demographic information of the patient, the name of the consulting hospital, the initial diagnostic impression, the diagnostic impression of a dermatologist, and the procedures, if any, performed by that dermatologist. RESULTS: A total of 429 patients were evaluated (53% men, 47% women) from July 2007 through June 2013. The most common diagnosis was infectious process (37%), followed by eczema (14%) and drug-induced skin reactions (12%). Seventeen percent (17%) of the cases for which consultations were sought were considered true dermatological emergencies. Forty-six percent of cases resulted in no diagnostic impression from the consulting physician. Of the cases that did result in diagnoses, these diagnoses were later changed by a dermatologist in 34% of the cases. CONCLUSION: This study suggests that the role of the dermatologist in the emergency department is very important. In addition, better education in the management of common skin disorders and the identification of true dermatological emergencies should be stressed during medical school and in residency training programs of specialties such as emergency medicine and those that offer primary care.

Early alterations of B cells in patients with septic shock.

INTRODUCTION: It has recently been proposed that B lymphocytes are involved in sepsis pathogenesis. The goal of this study is to investigate potential abnormalities in a subset distribution and activation of circulating B lymphocytes in patients with septic shock. METHODS: This observational prospective study was conducted in a medical-surgical ICU. All patients with septic shock were eligible for inclusion. B-cell phenotypes (CD19+CD69+, CD19+CD23+, CD19+CD5+, CD19+CD80, CD19+CD86+, CD19+CD40 and CD19+CD95+) were assessed by quantitative flow cytometry upon admission to the ICU and 3, 7, 14 and 28 d later. RESULTS: Fifty-two patients were included. Thirty-six healthy volunteers matched for age and sex were used as controls. The patients had lymphopenia that was maintained during 28 d of follow-up. In patients with septic shock who died, the percentage of CD19+CD23+ was lower during the 7 d of follow-up than it was in survival patients. Moreover, the percentage of CD80+ and CD95+ expression on B cells was higher in patients who died than in survivors. Receiver operating characteristic curve analysis showed that a CD19+CD23+ value of 64.6% at ICU admission enabled discrimination between survivors and nonsurvivors with a sensitivity of 90.9% and a specificity of 80.0% (P=0.0001). CONCLUSIONS: Patients with septic shock who survive and those who don't have different patterns of abnormalities in circulating B lymphocytes. At ICU admission, a low percentage of CD23+ and a high of CD80+ and CD95+ on B cells were associated with increased mortality of patients with septic shock. Moreover, a drop in circulating B cells persisted during 28 d of ICU follow-up.

The predictive role of early activation of natural killer cells in septic shock.

Recently, several studies about the role of natural killer (NK) cells in sepsis have been highlighted. In an earlier study, we characterized the abnormalities of circulating lymphocytes in 52 patients with septic shock during the first 28 days in the intensive care unit. Our results confirm and expand some previous reports. We found that patients who did not survive exhibited less NK cell (CD3-CD56⁺) depletion than survivors and that these NK cells expressed CD69⁺ and CD57⁺. These data demonstrate that NK cells are key participants in septic shock because patients who survived have more depletion and expressed less early activation and differentiation.

Accurate Enumeration of Apoptotic Cancer Cells Using Flow Cytometry.

The frequency of apoptotic cells in a given phenotypically defined population is usually calculated the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. The present chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.

Loss of surface antigens is a conserved feature of apoptotic lymphocytes from several mammalian species.

Human lymphocytes lose the expression of lineage antigens (LAgs) along apoptosis. Our aim was to extent our previous studies of LAg loss to rodent species, quantifying LAg expression on apoptotic murine lymphocytes using flow cytometry to measure alterations in cell permeability, phosphatidylserine exposure and caspase activation of CD3, CD5, CD4, CD8, CD19 and CD28 LAgs in highly purified lymphocyte populations. We found loss of expression by apoptotic cells of all LAgs studied in the three species analyzed except for CD3 antigen in mouse. We also found an early, rapid and dramatic reduction in the expression of CD28 by early apoptotic cells. We found several homologies across the three species in the kinetic of loss of several LAgs such as CD5, CD4 and CD28. These data suggest that the loss of expression of LAgs by apoptotic lymphocytes is a common and conserved feature of lymphocytes undergoing apoptosis in several mammalian species.

Mortality in patients with septic shock correlates with anti-inflammatory but not proinflammatory immunomodulatory molecules.

BACKGROUND: Mortality in patients with septic shock remains unacceptably high and the attempts to antagonize certain proinflammatory cytokines based on the results of animal model studies have failed to improve survival rates. The objective of this article is to examine the pro-/anti-inflammatory cytokine balance in patients with septic shock and its connection with mortality. METHODS: Serum levels of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin 1ß [IL-1ß], interferonγ [IFN-γ], and IL-6) and soluble cytokine antagonists (soluble TNF receptor I [sTNF-RI], sTNF-RII, and IL-1Ra) were determined on admission to the intensive care unit (ICU) and 3, 7, 14, and 28 days later in 52 patients with septic shock and in 36 healthy controls. Specific sandwich enzyme-linked immunosorbent assay (ELISA) was used for all determinations. RESULTS: Serum levels of most of the pro- and anti-inflammatory molecules examined (TNF-α, IL-6, sTNF-RI, sTNF-RII, and IL-1 receptor agonist [IL-1Ra]) were significantly elevated on admission and during the 28-day observation period in patients when compared to controls. Notably, the anti-inflammatory mediators sTNF-RI, sTNF-RII, and IL-1Ra were better predictors of mortality. Receiver-operating characteristic (ROC) analysis revealed that sTNF-RI or sTNF-RII concentrations over 2767 or 4619 pg/mL, respectively, determined a high risk of death (sensitivity: 100%-100%, specificity: 57.1%-71.4%, area under the curve [AUC] 0.759-0.841, respectively), whereas IL-1Ra concentrations below 7033 pg/mL determined a high probability of survival (sensitivity: 60%, specificity: 100%, AUC 0.724). In addition, IFN-γ levels were significantly higher in survivors than in controls during the initial 2 weeks of observation. CONCLUSIONS: Our data show that serum cytokine disturbance patterns have prognostic significance in patients with septic shock admitted to the ICU. The pattern, defined by an early response to continuously elevated anti-inflammatory cytokine serum levels, is associated with an enhanced risk of a fatal outcome for patients.

Human brucellosis is characterized by an intense Th1 profile associated with a defective monocyte function.

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1beta (IL-1beta), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-gamma, IL-2, and TNF-alpha, but not that of IL-4, by phorbol myristate-activated CD4(+) CD3(+) and CD8(+) CD3(+) T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-gamma levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.

Ancient genomes in South Patagonia reveal population movements associated with technological shifts and geography.

Archaeological research documents major technological shifts among people who have lived in the southern tip of South America (South Patagonia) during the last thirteen millennia, including the development of marine-based economies and changes in tools and raw materials. It has been proposed that movements of people spreading culture and technology propelled some of these shifts, but these hypotheses have not been tested with ancient DNA. Here we report genome-wide data from 20 ancient individuals, and co-analyze it with previously reported data. We reveal that immigration does not explain the appearance of marine adaptations in South Patagonia. We describe partial genetic continuity since ~6600 BP and two later gene flows correlated with technological changes: one between 4700-2000 BP that affected primarily marine-based groups, and a later one impacting all <2000 BP groups. From ~2200-1200 BP, mixture among neighbors resulted in a cline correlated to geographic ordering along the coast.

IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in active patients with multiple sclerosis.

Abnormal apoptosis has been reported in circulating T lymphocytes in patients with multiple sclerosis. The effects of 12 months of IFNbeta treatment in T and B lymphocyte spontaneous ex vivo apoptosis were studied in patients with MS. Peripheral blood mononuclear cells were obtained from 48 patients before and at 1, 6 and 12 months after treatment with IFNbeta. Spontaneous ex vivo apoptosis was quantified by four-color flow cytometry. A significant reduction and normalization of the percentage of apoptotic cells was found in all T lymphocyte subsets. B cell apoptosis values were unaffected by therapy; Relapses of the clinical activity of the disease were associated to transitory upturns of lymphocyte apoptosis. In conclusion, IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in MS. However, it is not clear if this reduction in spontaneous T lymphocyte apoptosis is due to direct effect of IFNbeta or secondary to decreased clinical and sub-clinical activity.

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis.

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.

Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients.

INTRODUCTION: Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications. METHODS: T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry. RESULTS: CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75. CONCLUSIONS: A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.

Evolution, systematics, and phylogeography of pleistocene horses in the new world: a molecular perspective.

The rich fossil record of horses has made them a classic example of evolutionary processes. However, while the overall picture of equid evolution is well known, the details are surprisingly poorly understood, especially for the later Pliocene and Pleistocene, c. 3 million to 0.01 million years (Ma) ago, and nowhere more so than in the Americas. There is no consensus on the number of equid species or even the number of lineages that existed in these continents. Likewise, the origin of the endemic South American genus Hippidion is unresolved, as is the phylogenetic position of the "stilt-legged" horses of North America. Using ancient DNA sequences, we show that, in contrast to current models based on morphology and a recent genetic study, Hippidion was phylogenetically close to the caballine (true) horses, with origins considerably more recent than the currently accepted date of c. 10 Ma. Furthermore, we show that stilt-legged horses, commonly regarded as Old World migrants related to the hemionid asses of Asia, were in fact an endemic North American lineage. Finally, our data suggest that there were fewer horse species in late Pleistocene North America than have been named on morphological grounds. Both caballine and stilt-legged lineages may each have comprised a single, wide-ranging species.

Flow cytometry enumeration of apoptotic cancer cells by apoptotic rate.

Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.

Treatment with AM3 restores defective T-cell function in COPD patients.

BACKGROUND: Lymphocyte alterations have been associated with an increased prevalence of acute respiratory infections in COPD patients. AM3 is an oral immunomodulator that normalizes the defective functions of peripheral blood natural killer and phagocytic cells in COPD patients and improves their health-related quality of life. OBJECTIVES: To characterize putative systemic abnormalities of the T-cell compartment in COPD patients, and to investigate whether AM3 can restore such abnormalities. DESIGN: The study was a randomized, prospective, double-blind, placebo-controlled trial in a cohort of COPD patients. The results were also compared to those of nonsmoker and ex-smoker healthy control subjects. SETTING: Outpatient departments of four hospitals. PATIENTS: Seventy COPD patients were randomized to receive either AM3 or a placebo orally for 90 consecutive days. Populations of 36 healthy nonsmokers and 36 healthy ex-smokers were used as control subjects. MEASUREMENTS: Peripheral blood mononuclear cell (PBMC) proliferation and production of interleukin (IL)-2, IL-4, IL-12p40, tumor necrosis factor-alpha, and interferon (IFN)-gamma proteins in response to the T-cell polyclonal mitogens were assessed at baseline and at the end of treatment. RESULTS: The proliferative response was significantly decreased in COPD patients. Decreased production of IFN-gamma was the only defect in the profiles of the cytokine measures, and was selectively observed in COPD patients, but not in nonsmoker and ex-smoker healthy control subjects. Treatment with AM3 significantly restored the PBMC proliferative response to polyclonal mitogens and significantly promoted stimulated IFN-gamma production in these patients. The normalization of these proliferative responses was not related to significant variations in the numbers of peripheral blood monocytes, CD3+, CD4+, CD8+ cells or of any major naïve/memory/activated T-cell subset. The increased IFN-gamma production in the AM3 study arm was associated with an increase in the mean of number of IFN-gamma molecules produced per CD8+ T cells. CONCLUSIONS: PBMCs of COPD patients showed clear functional T-lymphocyte abnormalities that are rescued by AM3 treatment.

Involvement of Bax, Bcl-2 and caspase 3 in hydroxyurea- or etoposide-induced apoptosis of mouse interleukin-3-dependent lymphoma cells.

BACKGROUND: The apoptosis action induced by hydroxyurea or etoposide in interleukin 3-dependent lymphoma cells (DA-1) was studied. MATERIALS AND METHODS: The conditions to study apoptosis of these cells were 17 hours of cell treatment with concentrations of 1.25 mM hydroxyurea or 100 microM etoposide using flow cytometry, fluorometry and immunoblots techniques. RESULTS: Time-dependent reductions of cell viability after these treatments were observed. Caspase 3 activity was highly activated in both cases. Chemical treatments or interleukin 3 withdrawal rendered level changes in Bax and Bcl-2 expression. CONCLUSION: These results support the implication of these factors in DA-1 cell apoptosis induced by these chemical treatments or interleukin 3 depletion.

Quantitation of apoptosis induction by etoposide or hydroxyurea in mouse interleukin 3-dependent lymphoma cells.

BACKGROUND: The apoptosis induction by etoposide or hydroxyurea in mouse interleukin 3-dependent lymphoma cells (DA-1) was studied. Treatments with 1.25 mM hydroxyurea and 100 microM etoposide for 17 hours were considered appropriate concentrations for such studies. MATERIALS AND METHODS: The viability of the cells was studied by propidium iodide exclusion. The presence of apoptotic bodies was observed, and DNA fragmentation of apoptotic cells was asessed by the observation of ladder patterns in agarose electrophoresis gels and an increase in the proportion of cells with subdiploid DNA content in cytometric studies. RESULTS: Alterations of membrane properties, DNA degradation and chromatin condensation highlighted a possible apoptotic process induced by these two antitumor agents. CONCLUSION: These studies have established conditions for examining apoptotic processes in interleukin 3-dependent lymphoma cells. This is of great interest for deeper analysis of the mechanisms involved in the apoptosis of these cells after interleukin 3 depletion or treatment with cytotoxic antitumor compounds.