Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Systemic distribution of viral antigen in alpacas persistently infected with bovine pestivirus.

Recently, confirmed occurrences of persistent bovine viral diarrhea virus (BVDV) infection in North American alpacas have raised concerns about the role of persistently infected (PI) alpacas in transmission of virus among herds, yet only limited pathological descriptions of persistent infections in alpacas have been reported. The objective of this study was to characterize BVDV antigen distribution in 10 PI alpacas of varying age and to compare viral antigen distribution and localization in tissues of PI alpacas with 5 PI calves of varying age. Ocular dysplasia was evident in 1 PI alpaca, constituting the first reported congenital ocular lesion in PI alpacas. Viral antigen was widely distributed in alpaca tissues and was prominent in neurons, endothelial cells, and vascular tunica media myocytes but had limited distribution in lymphoid tissues and moderate distribution in epithelium of several organ systems of alpacas. Macrophages in the alpaca gastrointestinal system submucosa and lymph node medullary sinuses often had prominent labeling. In addition, only 1 alpaca had antigen labeling in the bone marrow in contrast to PI cattle. Labeled cells in calf tissues were more widely distributed, occurring prominently in lymphoid and epithelial tissues. Common features of the 2 host species were widespread antigen labeling and absence of lymphoid depletion.

Long-term clinicopathological characteristics of alpacas naturally infected with bovine viral diarrhea virus type Ib.

BACKGROUND: Substantial bovine viral diarrhea virus (BVDV)-related production losses in North American alpaca herds have been associated with BVDV type Ib infection. OBJECTIVES: To classify and differentiate the long-term clinicopathological characteristics of BVDV type Ib infection of alpaca crias, after natural virus exposure. We hypothesized that persistently infected (PI) alpacas specifically demonstrate growth retardation, clinicopathological evidence of opportunistic infections, and early mortality. ANIMALS: Thirty-five crias naturally exposed to BVDV (18 acute, 3 chronic, 14 PIs), and 19 healthy cohort controls of 5 northeastern alpaca farms were prospectively evaluated over 2 years (September 2005-September 2008). METHODS: Observational cohort-control study. RESULTS: Chronically (viremia >3 weeks) and PI crias demonstrated significantly lower birth weights, decreased growth rates, anemia, and monocytosis compared with control animals. Common clinical problems of PI alpacas included chronic wasting, diarrhea, and respiratory disease. Median survival of PI alpacas that died was 177 days (interquartile range, 555) with a case fatality rate of 50% within 6 months of life. Transplacental infection was confirmed in 82% (9/11) of pregnant females on 1 farm, resulting in the birth of 7 PI crias (7/10 deliveries; 1 animal was aborted). Mean gestation at the beginning and end of BVDV exposure was 64 and 114 days, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Natural BVDV type 1b infection during early pregnancy resulted in a high incidence of PI offspring. Although PI alpacas may have distinct clinical characteristics, verification of persistent viremia in the absence of endogenous, neutralizing antibodies is essential to differentiate persistent from chronic infection.

Persistent bovine viral diarrhoea virus infection in US beef herds.

In the summer of 1996, we screened 18,931 calves in 128 beef herds located in five US states for persistent bovine viral diarrhea virus (BVDV) infection. Of these, 76 herds were randomly selected from the client database of collaborating veterinary practices, and 52 herds were suspected by the collaborating veterinarians to have BVDV infection based on history or clinical signs. Serum was obtained from each calf in the cooperating herds prior to 4 months of age and tested for the presence of BVDV by microtiter virus isolation. Information about each of the herds (including management practices, vaccination history, and breeding- and calving-season production measures) were collected by the collaborating veterinarians using standardized questionnaires. A total of 56 BVDV-positive calves in 13 herds were identified on initial screening. Ten (19%) of the BVDV-suspect herds and three (4%) of the randomly selected herds had > or = 1 BVDV-positive calf at initial screening. Multiple BVDV-positive calves were identified in 10 of those 13 herds. Follow-up information was obtained for 54 of the 56 positive calves. Ten out of 54 (18%) died prior to weaning, and 1 (2%) was sold because of unusually poor growth. Thirty-three out of 54 (61%) of the initially positive calves remained BVDV positive at 6 months of age - confirming persistent-infection (PI) status. Dams of 45 of the 56 positive calves were tested, with 3 (7%) identified as positive - indicating most PI calves were products of acute dam infection during gestation. The proportion of cows that were pregnant at the fall 1995 pregnancy examination was 5% lower in herds with PI calves born during the 1996 calving season than in randomly selected herds without PI calves. Most of the calves we identified with persistent BVDV infections survived to weaning, and could provide a constant source of virus to the herd throughout the breeding season and early gestation.

In situ hybridization detection of bovine respiratory syncytial virus in the lung of experimentally infected lambs.

We studied the distribution of bovine respiratory syncytial virus (BRSV) RNA in lungs of experimentally inoculated lambs by in situ hybridization at different times postinoculation. The probe used for in situ hybridization was prepared by reverse transcription of BRSV RNA, followed by polymerase chain reaction (PCR) amplification of the cDNA. Twenty-five Merino lambs of both sexes with a live weight of 17 +/- 3 kg received an intratracheal inoculation of 20 ml saline solution containing 1.26 X 10(6) TCID50 BRSV (strain NMK7)/ml. Lambs were slaughtered 1, 3, 7, 11, and 15 days postinoculation (PID). Bronchial and bronchiolar epithelial cells were positive for BRSV nucleic acid by ISH at 1, 3, 7, and 11 PID. However, alveolar epithelial cells contained positive cells at 1, 3, and 7 PID. Cells containing viral RNA were detected from 1 to 11 PID in exudate within bronchial and bronchiolar lumina and from 3 to 7 PID in alveolar exudates. Positive hybridization signals were identified in interstitial mononuclear cells and in bronchi-associated lymphoid tissue from 3 to 11 PID. Mononuclear cells were located in peribronchiolar tissue and interalveolar septa. The highest signal intensity in positive cells was observed at 3 and 7 PID, coinciding with the most important histopathological findings.

Immunogenicity and protective efficacy of a gE, gG and US2 gene-deleted bovine herpesvirus-1 (BHV-1) vaccine.

The efficacy and safety of a gene-deleted bovine herpesvirus-1 (BHV-1) vaccine was determined in a bovine herpesvirus challenge trial in calves. Three different doses of the vaccine were administered intramuscularly at 10(5), 10(6) and 10(7) PFU/ml and compared to a commercial vaccine and non vaccinated control calves. Challenge was performed by intranasal aerosolization with the Cooper strain of BHV-1 (3 x 10(4) PFU/ml). The non-vaccinated calves shed significantly (P < 0.05) more virus than all other groups on days 4, 8 and 10 post challenge. By day 14 post challenge, antibody titers for BHV-1 of calves vaccinated with 10(7) PFU/ml were significantly (P < 0.05) higher than the commercial or non-vaccinated calves. Clinical scores of non-vaccinated calves were significantly (P < 0.05) higher than all other groups on days 4-14 post challenge. With both radioimmunoprecipitation and competitive enzyme-linked immunosorbent assays (C-ELISA), calves in the gene-deleted vaccine groups mounted comparable specific responses against gB, gC and gD post vaccination as calves in the commercial vaccine group, but in a dose dependent manner. These data suggest that the gene-deleted BHV-1 vaccine tested may be used as an effective vaccine in controlling BHV-1 infections.

Experimental infection of calves with bovine viral diarrhea virus genotype II (NY-93).

To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.

Alteration of leukocyte populations in calves concurrently infected with bovine respiratory syncytial virus and bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection altered leukocyte populations in calves that were reflected by depression of T, BoCD4+, and BoCD8+ lymphocytes in the thymus and depression of B lymphocytes in Peyer's patches (PP). The present study was based on mononuclear leukocyte preparations from eighteen 9- to 12-month-old crossbred calves that were each exposed to either bovine respiratory syncytial virus (BRSV), BVDV, or BRSV and BVDV concurrently, or served as mock-infected controls. Peripheral blood leukocytes were collected on postinfection days (PID) 0, 2, 4, 6, and 8, and cell populations from thymus, spleen, mesenteric lymph node, and PP were collected at necropsy on PID 9. The leukocytes were analyzed using flow cytometry for lymphocyte subpopulations expressing antigens specific for BoCD2, BoCD4, BoCD8, BoWC1, lambda light chain of bovine immunoglobulin, BoCD11b and major histocompatibility complex (MHC) class II. Concurrent BRSV and BVDV infections caused exaggerated alterations in leukocyte populations with a greater percentage of T-lymphocytes harvested from the PP. Alterations in the leukocyte populations in lymphatic tissues and in peripheral circulation due to BVDV infection may be an important mechanism for causation of clinically severe diseases of the respiratory and digestive tracts during concurrent BRSV and BVDV infections.

Effect of concurrent experimentally induced bovine respiratory syncytial virus and bovine viral diarrhea virus infection on respiratory tract and enteric diseases in calves.

OBJECTIVE: To compare experimentally induced concurrent bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) infection with single virus infection. ANIMALS: 9- to 12-month-old calves. PROCEDURE: Calves were allotted to 4 groups: 1, mock-infected control (n = 3); 2, BRSV infected (5); 3, BVDV infected (5); and 4, concurrent BRSV and BVDV infected (5). Total and differential WBC counting was done. Concentration and duration of BVDV in nasal secretions and serum, and duration of BRSV in nasal secretions were determined. Concentration of BVDV in various tissues was determined, and isolation of BRSV from lung tissue was attempted. Histologic examination and immunohistochemical analysis were done to detect lesions and distribution of viral antigens, respectively. RESULTS: Calves with concurrent infection developed more severe clinical signs of disease (fever and diarrhea), leukopenia, and more severe lesions. They also shed virus from nasal secretions in greater concentration and for longer duration, and BRSV was isolated from their lungs. Calves with concurrent infection also had more extensive lung lesions. Alimentary epithelial necrosis and severe lymphoid depletion were associated with BVDV infection in calves with or without concurrent BRSV infection. BVDV antigen in lymphatic tissue was detected in stromal cells only. CONCLUSIONS: Concurrent infection with BRSV and BVDV resulted in more severe respiratory tract and enteric disease than did infection with either virus alone, possibly indicating synergistic effect between the viruses. BVDV's role in causing respiratory tract disease is attributable, indirectly, to effects on the host's immune system, not to infection of the lungs.

Prevalence of morphologic defects in spermatozoa from beef bulls.

OBJECTIVE: To determine the overall prevalence of morphologic defects in spermatozoa from beef bulls and to determine whether prevalence varies with the age of the bull. DESIGN: Cross-sectional observational study. ANIMALS: 2,497 beef bulls that were evaluated for breeding soundness in 1994 by 29 practicing veterinarians in a 5-state geographic region. PROCEDURE: Slides of spermatozoa from each bull were made and submitted by practicing veterinarians for morphologic evaluation. One hundred spermatozoa per slide were examined, and each was classified as having 1 of 9 morphologic defects or as normal. RESULTS: 63% of bulls evaluated were 10 to 12 months old, and 20% were 13 to 18 months old. A mean of 70.6% of spermatozoa was classified as normal. Most common defects were proximal droplets (8.4%), distal midpiece reflexes (6.7%), separated heads (5.5%), and distal droplets (3.8%). Other defects were seen < 2% of the time. Bulls 10 to 12 months of age had a higher prevalence of proximal and distal droplet defects than older bulls. CLINICAL IMPLICATIONS: Practitioners conducting breeding soundness evaluations in beef bulls must be aware of common spermatozoal defects. Bulls that are evaluated at a young age will have more defects than older bulls and should be reevaluated, particularly for those defects for which prevalence decreases with age.

Virulence markers in the 5' untranslated region of genotype 2 bovine viral diarrhea virus isolates.

Virulence markers to distinguish high from low virulence bovine viral diarrhea virus genotype 2 isolates have not been previously reported. The objective of this study was to identify virulence markers by evaluating the primary and secondary structures of the 5'-untranslated region of low and high virulence bovine viral diarrhea virus genotype 2 isolates. The nucleotide sequences of the entire 5'-untranslated region mRNA of eight bovine viral diarrhea virus genotype 2 isolates, four of high virulence and four of low virulence, and two genotype 1 reference isolates were determined using a polymerase chain reaction and a 5' Rapid Amplification of cDNA Ends System. Two nucleotide substitutions were identified in the internal ribosomal entry site that distinguished the high virulence from the low virulence genotype 2 isolates. The low virulence isolates had a cytosine at position 219, whereas the high virulence isolates had a uracil. At position 278, a uracil or cytosine was found in the low and high virulence groups, respectively. The substituted bases are virulence markers that were used to identify bovine viral diarrhea virus genotype 2 isolates of high virulence.

Safety assessment of enzyme-containing personal cleansing products: exposure characterization and development of IgE antibody to enzymes after a 6-month use test.

BACKGROUND: Enzyme-containing personal cleansing products were being considered for the consumer market. Although enzymes have been marketed safely for many years as ingredients in laundry products, their use in a personal cleansing application represented a new type of exposure for consumers that was not supported by the historical safety data. An exposure assessment and additional safety data would be needed before marketing to ensure consumer safety. OBJECTIVE: The work in this paper was designed to evaluate the potential for inhalation exposure to the enzyme during use of this new product while showering. Then a clinical trial was conducted to determine whether or not the level, duration, and routes of exposure encountered during use of this product would induce a Type I sensitization response to the enzyme. METHODS: Exposure was assessed during normal showering activities by collecting air samples with both high volume and personal samplers and quantitating enzyme levels with an ELISA. To assess the potential for sensitization, panelists were asked to use a prototype protease-containing bar product for all personal cleansing tasks and to keep a use diary reporting any associated symptoms. Physical and dermatologic examinations and skin prick tests with enzyme were conducted before the test commenced and at 2-month intervals. RESULTS: Exposure assessment results showed that airborne enzyme levels were primarily dependent on the concentration of the enzyme in the personal cleansing product. Mean values for total airborne enzyme protein ranged from 5.7 to 11.8 ng/m3 when enzyme concentration, time of use, and measurement technique remained constant. After 6 months of at-home product use, four of 61 test subjects using the enzyme-containing bar had positive skin prick test responses when tested with the enzyme. The skin prick test data were supplemented with serologic analyses, which detected IgE specific for the protease enzyme. None of these subjects showed any clinical symptoms indicative of allergic reaction. CONCLUSION: The ability of enzymes to induce development of allergic antibodies in this study led to the conclusion that this prototype enzyme-containing personal cleansing bar would represent an inappropriate use of enzymes in a consumer product application. The likelihood of both induction of an immunologic response and subsequent elicitation of allergy symptoms in a small but significant fraction of the user population was high. This finding resulted in the decision to halt further development of this prototype.

Percutaneous penetration of N-nitroso-N-methyldodecylamine through human skin in vitro: application from cosmetic vehicles.

The human skin penetration of N-nitroso-N-methyldodecylamine (NDOMA) from isopropyl myristate (IPM) and two vehicles representative of cosmetic/personal care formulations was determined in vitro. When applied as an infinite dose in IPM (1 microgram/microliter) the average total absorption over 48 hr was 0.10 +/- 0.01% of the applied dose (all data are expressed as means +/- SE). When applied as a finite dose in a representative oil-in-water emulsion formulation the average total absorption over 48 hr was 4.66 +/- 0.76% of the applied dose. When applied as a finite dose in a representative shampoo formulation for 10 min, followed by rinsing (to represent in-use exposure conditions), the average total absorption over 48 hr was 0.75 +/- 0.17% of the applied dose. Approximately 72% of the NDOMA in the applied shampoo formulation was removed by rinsing. The overall data indicated that NDOMA could penetrate the skin but that penetration was low. The rate and extent of absorption, however, could be affected by differences in the vehicle of application, time of exposure and whether the formulation is (and the conditions are designed to mimic) a rinse-off or leave-on product.

Sequence conservation in the attachment glycoprotein and antigenic diversity among bovine respiratory syncytial virus isolates.

Partial nucleotide sequences were determined from the coding regions of the attachment glycoprotein (G) mRNAS of eight isolates of bovine respiratory syncytial virus (BRSV). The antigenic characteristics of 18 field and reference isolates were analyzed using the reactivity patterns of monoclonal antibodies (MAbs) directed against the human respiratory syncytial virus (HRSV) and BRSV G. fusion protein (F), nucleoprotein (N), and phosphoprotein (P), by radioimmunoprecipitation and immunofluorescence assays. The MAb reaction patterns demonstrated some random antigenic differences among the isolates, but for the most part were cross-reactive to the viral protein epitopes, especially on the F protein. Structural differences in the F and P proteins were observed among BRSV isolates; the P protein migrated at three different apparent molecular weights on PAGE gels. Antigenic and structural variation occurs among isolates, however, the structural differences in the P protein did not correlate with the antigenic differences among the F, N and P proteins. The G mRNA nucleotide sequence identities were high, ranging from 94.1 to 99.9%, and the predicted amino acid sequence identities ranged from 89.9 to 99.6%. Variance was due to substitution point mutations. The G protein ectodomains contained areas of sequence divergence flanking a highly conserved region, with four cysteine residues, which is analogous to the putative HRSV receptor binding domain. The high sequence and amino acid identities and random antigenic diversity among the isolates indicates that the BRSV isolates analyzed belong in a monophyletic group.

Virulence of feline Chlamydia psittaci in mice is not a function of the major outer membrane protein (MOMP).

The virulent strain of feline Chlamydia psittaci, the Cello strain, produces conjunctivitis and upper respiratory disease in cats. This same strain produces a lethal disease in mice when inoculated intraperitoneally (i.p.). In this study we have shown that the Baker strain of feline C. psittaci is attenuated in the mouse model system. Intraperitoneal inoculation of mice with the Baker strain produced no disease but did stimulate an immune response that protected the mice from subsequent produced i.p. inoculation with the virulent Cello strain. To determine if the difference between these two strains was in the major outer membrane protein (MOMP), the omp1 gene which codes for this protein was sequenced for both the Baker and Cello strains. The MOMP was chosen to study because in Chlamydia trachomatis this protein has been shown to contain neutralizing epitopes and has been shown to play a role in cell attachment. These functions make it a likely structural component capable of mutating and causing altered cell tropism and virulence. The DNA sequence of the omp1 was determined by amplifying the gene with PCR, cloning the PCR product into the pCR-II cloning vector and determining the DNA sequence of the inserted gene using primers to sites in the plasmid vector. From the DNA sequence, the deduced amino acid sequence of MOMP was determined for both the attenuated Baker and the virulent Cello strains of feline C. Psittaci. The results indicated that the omp1 gene of both strains contained 1179 base pairs which coded for a protein 392 amino acids. The DNA sequences of the omp1 gene of the two strains differed by only two base pairs which resulted in two amino acid changes in the MOMP. The Baker strain had a serine instead of a tryptophan at amino acid 7 and a tyrosine instead of an aspartic acid at amino acid 125 of the uncleaved protein. Neither amino acid change was in an area of the MOMP which could logically account for the difference in biological activity. Amino acid 7 was in the leader sequence which is cleaved from the authentic MOMP and is not present in the infectious elementary body. Amino acid 125 was in a conserved hydrophobic area of one of the constant regions of the protein. A change at this location was not likely surface exposed and thus could not affect cell adhesion, tissue tropism or neutralizing epitopes. Therefore, the differences in the primary structure of the MOMP from the Baker and Cello strains of feline C. psittaci could not account for the attenuation of the Baker strain for mice. The molecular basis of their difference is yet to be determined.

Distribution of virus and viral antigen in specific pathogen-free calves following inoculation with noncytopathic bovine viral diarrhea virus.

Gross and microscopic lesions and distribution of virus were studied in specific pathogen-free calves (SPF) 10 days post-inoculation (PI) with bovine viral diarrhea virus (BVDV). To investigate possible differences in tissue tropism between BCDV isolates, two clinically and antigenically different noncytopathic (ncp) isolates of BVDV were compared in the study. Four calves were exposed to noncytopathic (ncp) BVDV 7937, and four to ncp-BVDV 126. Two additional calves that were not exposed to virus served as controls. Both ncp-BVDV 7937 and ncp-BVDV 126 induced mild disease characterized by variable fever and anorexia. Lymphoid depletion was evident in Peyer's patch of four calves and the thymus of two calves exposed to BVDV. Differences between these isolates in the distribution of BVDV or BVDV antigen in tissues of inoculated calves were not found. High concentrations of BVDV and BVDV-specific antigen were detected in the thymus, Peyer's patch, and mesenteric lymph node of all exposed calves. BVDV was shown to infect cells of the bone marrow without causing microscopic lesions. High concentrations of BVDV were recovered from the bone marrow of all calves exposed to BVDV and BVDV-specific antigen was demonstrated at this location in six of these calves. Platelet counts of calves exposed to BVDV were significantly reduced during infection, which resulted in thrombocytopenia in one calf. Focal areas of necrosis were observed in squamous epithelial cells of the tonsil and ruminal mucosa. BVDV-specific antigen was found in and adjacent to these foci. Calves exposed to ncp-BVDV 7937 had slightly more severe clinical signs than those exposed to ncp-BVDV 126.

Percutaneous penetration of dimethylnitrosamine through human skin in vitro: application from cosmetic vehicles.

Human skin penetration of N-dimethylnitrosamine (DMN) from three vehicles has been determined in vitro. When applied as an infinite dose in isopropyl myristate (IPM, 1 microgram/microliter) the average total absorption over 48 hr was 2.6 +/- 1.2% of the applied dose (all data presented are expressed as means +/- standard errors). When applied as a finite dose in a representative oil-in-water emulsion vehicle the average total absorption over 48 hr was 4.0 +/- 0.3% of the applied dose. When applied as a finite dose in a representative shampoo vehicle for 10 min followed by rinsing (i.e. to represent in-use exposure conditions) the average total absorption over 48 hr was 1.1 +/- 0.1% of the applied dose. Approximately 72% of the DMN in the applied shampoo vehicle was removed by rinsing. There was considerable evaporative loss of DMN from the IPM and oil-in-water emulsion vehicles, such that absorption was complete within 3 hr of application. The overall data indicate that DMN can penetrate the skin rapidly but that in practice the amount actually available for penetration is significantly reduced by high permeant volatility. In contrast, application of N-nitrosodiethanolamine (NDELA) at a concentration of 1 microgram/microliter as an infinite dose generated an average total absorption over 48 hr of 23.6 +/- 6.4%, representing a total flux of 103.9 +/- 28.4 micrograms/cm2. In the case of NDELA, no evaporative loss was evident.