Analysis of covariance–adjusted bone data for clinic site, age, and limb length (model 1) and further adjusted for body weight (model 2) were used to compare data between participants with (n = 190) and without (n = 981) T2DM. At both the distal tibia and radius, patients with T2DM had greater bone vBMD  and a smaller bone area . The higher vBMD compensated for lower bone area, resulting in no differences in estimated compressive bone strength at the distal trabecular bone regions. At the mostly cortical bone midshaft sites of the radius and tibia, men with T2DM had lower ToA , resulting in lower bone bending strength at both sites after adjusting for body weight  despite the lack of difference in cortical vBMD at these sites. These data demonstrate that older men with T2DM have bone strength that is low relative to body weight at the cortical-rich midshaft of the radius despite no difference in cortical vBMD. © 2010 American Society for Bone and Mineral ResearchThe effects of type 2 diabetes mellitus (T2DM) on bone volumetric density, bone geometry, and estimates of bone strength are not well established. We used peripheral quantitative computed tomography (pQCT) to compare tibial and radial bone volumetric density (vBMD, mg/cm Observational cohort studies have found that type 2 diabetes mellitus (T2DM) is associated with a 50% to 80% increased risk of hip fracture, as well as a 30% to 70% increased risk of fracture of the proximal humerus and foot.–3 Althou2) assessed by dual x-ray absorptiometry (DXA) in patients with T2DM compared with healthy controls, even after accounting for larger body size among diabetics.(strength can be reduced even when there are no changes (or even an increase) in aBMD because of geometric changes., but bo changes.A 3D imaging technique, peripheral quantitative computed tomography (pQCT), has allowed for assessment of volumetric density of both cortical and trabecular compartments, as well as structural estimates of bone strength derived from cross-sectional geometry.,15 This The primary objective of this study was to examine the association between T2DM and bone volumetric density, geometry, and estimates of bone strength in community-dwelling older men. We hypothesized that men with T2DM would have lower bone strength relative to their body weight, particularly in highly trabecular regions such as the distal radius and distal tibia.From March 2000 through April 2002, 5995 men who were at least 65 years of age were enrolled in the baseline examination of the prospective Osteoporotic Fractures in Men (MrOS) study. Men wereMen who returned for their second exam an average of 4.7 ± 0.4 years later were invited to participate in an ancillary study involving pQCT. Of the 1550 men who attended the second exam at the Pittsburgh and Minneapolis study sites, 1171 (76%) completed the clinic visit and agreed to participate in the pQCT ancillary study and are included in this analysis. This ancillary study was approved by the institutional review boards at the Minneapolis and Pittsburgh sites. All participants in the ancillary study signed a written informed consent for the pQCT portion of the study.Peripheral quantitative computed tomography (pQCT) was used to obtain slices (2.3 ± 0.2 mm) at the 4% and 66% sites of the left tibia and at 4% and 33% of the nondominant forearm (radius). Slices are taken as a percentage of limb length from the distal end of the relevant bone. The XCT 2000 device  and the XCT-3000 device  were used to obtain the scans in Pittsburgh and Minneapolis, respectively. The only difference between the 2000 and 3000 scanners is the gantry size. The same acquisition and analysis software was used to analyze scans at both sites. We performed a precision study using a European forearm phantom scanned three times at each site at 200, 100, and 50 mg/cc, respectively. Values on the two instruments were similar and within less tha 0.5% for total area at all mg/cc values and from 0.5% to 1.0% for total density.3) and Peel mode 1 (45% area) were used. Distal sites were assessed for total bone cross-sectional area  and total density . Bone strength index  was calculated as (ToA × ToD2)/1,000,000 as an index of bone compressive strength. At the more cortical 33% radius and 66% tibia sites, we used Contour mode 2 (169 mg/cm3) to determine whole bone properties and Cortmode 1 (710 mg/cm3) for cortical bone properties. A threshold of 280 mg/cm3 was used to determine the polar strength strain index (SSIp). At these cortical sites, we assessed total bone cross-sectional area , cortical area , and cortical density . Polar strength strain index  and section modulus (mm3) were calculated as estimates of bone bending strength,(2) was measured (at the 66% tibia only). SSIp is a “density-weighted” section modulus value, whereas section modulus includes only geometric properties. An anthropomorphic phantom was scanned daily for quality assurance at both sites.Voxel size was 0.5 mm, and the scan speed was 25 mm/s. The anatomic reference line  was determined by acquisition of a 30 mm planar scout view of the joint line. Data were analyzed according to the manufacturer's specifications. At the trabecular 4% sites, Contour mode 2 , and total femur aBMD and body composition  were measured at the second exam using a fan-beam dual-energy X-ray absorptiometry . Standardized procedures for participant positioning and scan analysis were executed for all scans. All DXA operators were centrally certified on the basis of an evaluation of scanning and analysis techniques. Cross-calibration studies performed prior to the baseline MrOS visit found no linear differences across scanners, and the maximum percentage difference in mean total spine BMD between scanners was 1.4%. LongitudInformation on demographics, medical and family history, and lifestyle were obtained by questionnaire and interview by trained clinical staff at each site. Information regarding age and race (white/nonwhite) was collected.Presence or absence of T2DM was assessed by a self-report of physician diagnosis of diabetes or current use (at exam 2) of diabetes prescription medications, including hypoglycemics, sulfonylureas, or insulin. Fasting glucose and A1C assays were not available at exam 2.2). Prior to pQCT measurements, tibia and forearm length were measured to the nearest millimeter with an anthropometric tape measure. Tibia length was measured from the tibial plateau to the medial malleolus, and forearm length was measured from the ulnar styloid process to the olecranon process. The mean of two measurements for each variable was used for the analysis.Weight was measured in indoor clothing without shoes using a calibrated balance beam or digital scale. Height was measured using a Harpenden stadiometer , and body mass index was calculated  and ability to stand from a chair without using arms (yes/no) also were recorded. Grip strength was measured twice by a handheld dynamometer (Jamar) in both the left and right arms. The averFor lifestyle factors, men were classified into current, past, or never smokers using data collected from their current and baseline exams. Physical activity was measured by computing the Physical Activity Summary Scale for the Elderly (PASE).Differences in characteristics according to T2DM status were compared using analysis of variance for normally distributed continuous data and chi-square tests for categorical data.Bone measures were expressed as continuous variables using linear regression models, and the least-squared means procedure was used to estimate the mean  for each bone parameter by T2DM status (yes/no). Data are presented for two models first adjusted for age groups , race and tibia/radial length (model 1), and an additional model with further adjustment for body weight (model 2). Additional models were run substituting either lean mass or fat mass or body mass index instead of body weight, and results were similar (data not shown). Therefore, only results from models 1 and 2 are presented.n = 540) or Pittsburgh (n = 631) MrOs sites, 16% (n = 190) of whom had T2DM. A majority of men in both groups were Caucasian (98%), with 1% of participants being African American and 1% multiracial/other. Men with and without T2DM were similar in age, height, and tibia and radius length (p < .001) and had a significantly higher average BMI . Diabetic participants also had higher absolute levels of total body lean and fat mass (see p < .001). Men with diabetes also were significantly less active, had a lower grip strength, and took longer to complete the chair stand .A total of 1171 participants had pQCT scans and complete visit data at either the Minnesota (s length . As expemass see  and a sltand see . Slightltand see  men withp = .025) and for total area after adjusting for weight (p = .007). The slightly greater density in diabetics compensated for lower bone area, so there was no difference in estimates of compressive bone strength (BSI) between groups at this site after adjusting for body weight  and model 2 (adding body weight to the model) are presented in p = .031). Cortical area was similar between groups both before and after adjusting for body weight. Cortical bone density was not different between groups at this site in either model. The smaller total bone area translated to a lower bone bending strength  after adjusting for body weight. SSIp also tended to be lower in men with diabetes after adjusting for body weight (−2.5%) but was not significantly different (p = .106). Prior to adjusting for body weight, however, there was no difference in section modulus (p = .819) or SSIp between groups (p = .636). Diabetic participants also had significantly larger muscle CSA , although the muscle size was appropriate for their higher body weight .Participants with diabetes also tended to have smaller total bone area (−1% to −2%) at the cortical 66% site of the tibia, which was significantly different only after adjusting for body weight (p = .284). Differences in volumetric density were significant both before (p = .012) and after (p = .029) adjusting for body weight, whereas total area differences reached significance only after adjusting for body weight (p = .003). At the cortical 33% site, bone strength again was significantly lower relative to body weight  in men with T2DM because total  and cortical  bone areas were significantly smaller after adjusting for body weight. Cortical bone density was not different between groups in either model. Again, bone strength was not different prior to adjusting for body weight  among men with T2DM translated to lower bone bending strength (relative to body weight) in cortical regions of the tibia and radius.Epidemiologic studies suggest that patients with T2DM are at increased risk of hip, foot, and ankle fractures despite having consistently higher bone mineral density measured by DXA.,9,25 Theboth the load on the bone during the fall and the strength of bone itself. have conflicting effects on bone metabolism. For examat model.,18increased bone density and smaller bone area at the trabecular sites. Data from Krakauer and colleagues Low bone turnover, coupled with impaired bone formation, may explain our findings of low bone area and greater vBMD in this population. However, pQCT has limited resolution and is unable to assess trabecular thickness or connectivity. Studies using higher-resolution instruments are needed to further explore these findings.At the distal trabecular regions of the tibia and radius, bone density was significantly higher in patients with diabetes. We had hypothesized that trabecular bone density would be compromised owing to data showing that, in animals, T2DM primarily impacts trabecular bone.,18 Owingolleagues may helpolleagues A lower olleagues  that we did not measure may have influenced our findings. For instance, the majority of our participants were on some form of diabetes medication. While metformin may have a positive effect on bone, adverse There are several strengths to our study, including the unique focus on older men. Although it is estimated that one in four men will experience an osteoporotic fracture in their lifetime, few studies have focused on risk factors for osteoporosis and fracture in older men. Another strength of the current study was the assessment of volumetric BMD, bone geometry, and structural strength estimates that allowed for characterization of the underpinnings of bone strength differences in older men with and without T2DM. There are, however, several factors we could not address adequately that may have influenced our results. In particular, the duration or severity of diabetes were not assessed. We did not have information on the disease duration and were unable to adequately assess disease severity owing to a lack of hemoglobin A1C outcomes at exam 2. In our sample, there were no clear differences between men taking insulin versus other hypoglycemics (data not shown). However, we had a relatively small sample size in each strata, with only 27 men taking insulin. In addition, low testosterone and growth hormone levels are associated with T2DM and may influence bone stiffness. As with While past studies have demonstrated greater aBMD in T2DM, the current study, using pQCT, showed that type 2 diabetics had low bone strength for body weight at predominantly cortical sites. These findings, combined with the propensity to fall, may help to explain the increased risk of fracture in patients with type 2 diabetes.


Four of the six animals died or were euthanized due to their moribund conditions. Both animals that received the lowest exposure doses survived to the end of the observation period. The inhalation LD50 was determined to be approximately 7.8 × 104 pfu inhaled. These data demonstrate that an inhalation MPXV infection model has been developed in the cynomolgus macaque with disease course and lethal dose similar to previously published data.An inhalation exposure system was characterized to deliver aerosolized monkeypox virus (MPXV), and a non-human primate (NHP) inhalation monkeypox model was developed in cynomolgus macaques. A head-only aerosol exposure system was characterized, and two sampling methods were evaluated: liquid impingement via an impinger and impaction via a gelatin filter. The aerosol concentrations obtained with the gelatin filter and impinger were virtually identical, indicating that either method is acceptable for sampling aerosols containing MPXV. The mass median aerodynamic diameter (MMAD) for individual aerosol tests in the aerosol system characterization and the NHP study ranged from 1.08 to 1.15 μm, indicating that the aerosol particles were of a sufficient size to reach the alveoli. Six cynomolgus macaques  were used on study. The animals were aerosol exposed with MPXV and received doses between 2.51 × 10 Poxviridae family, is a highly contagious and debilitating systemic disease that is lethal to approximately 25% of its victims  smallpox model likely will be required. Although the variola virus can infect NHPs, it does not result in a lethal systemic disease. Furthermore, the variola virus exists in only two repositories in the world and is not available for broad testing of therapeutics and vaccines.Monkeypox virus (MPXV) is also a member of the Orthopoxvirus genus and presents a surrogate model for characterizing antivirals and vaccines to treat or prevent smallpox. Humans can be infected with MPXV, and the clinical presentation is similar to classic smallpox  monkeys  were obtained from a USDA certified vendor . The NHPs were free of obvious clinical signs of illness or malformations and tested negative for simian immunodeficiency virus (SIV), simian T-lymphotrophic virus 1 (STLV-1), macacacine herpesvirus 1 (herpes B virus), simian retrovirus 1 (SRV1) and SRV2, and for poxvirus antibodies prior to placement on study. The research was conducted in compliance with the Animal Welfare Act and followed the principles in the Guide for the Care and Use of Laboratory Animals from the National Research Council. Additionally, the research was conducted following an Institutional Animal Care and Use Committee (IACUC) approved protocol. The institution where the research was conducted is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All MPXV exposed animals were housed within a Biosafety Level 3 (BSL-3) containment laboratory.Six specific pathogen free MPXV (Zaire V79-I-005) strain was obtained from the Biodefense and Emerging Infections Research Resource Repository (BEI Resources). Monkeypox viral stocks were prepared in T-150 Vero cell flasks . The virus was diluted in Eagle's minimal essential medium  with 5% fetal bovine serum  without antibiotics to varying concentrations for use in a nebulizer for aerosol exposure. A virus plaque assay was used to quantitate the virus in the challenge material, impinger samples, and gelatin filter samples.2, rocking plates approximately every 15 min. After one hour, one mL of complete media were added to each well and incubated for 4 days at 37°C. On day 3, media were removed from the plates and stained with 0.5–1 mL crystal violet solution for 15 min and rinsed with PBS. Once the cells were dry, the number of plaques were counted.MPXV was enumerated in complete Eagles Minimum Essential Media (EMEM) with 0.1% gentamycin sulfate and 5% FBS and warmed to 37°C. Plaque assays were carried out on plates containing Vero 76 cells at ≥90% confluency obtained from Diagnostic Hybids Inc, . For each sample, one hundred mL of each dilution was added to each well of a twelve-well plate. Plates were incubated for 1 hour in at 37°C at 5% CO3, 6.94 × 104, and 7.19 × 105 pfu/mL) were aerosolized in duplicate for 10 min. One day of testing was performed to characterize a head-only aerosol exposure system, and two sampling methods were evaluated. The aerosol system was operated as described previously  determination (aerosol conc/nebulizer conc). Three starting nebulizer concentrations  at approximately 3 mg/kg. Each NHP was placed into a biological safety cabinet III (BSCIII) and loaded into a plethysmograph. Body plethysmography was performed real-time using Buxco Biosystems XA  while the animal's head was placed within an exposure chamber. Air was supplied to the system by an in-house air system filtered through a carbon filter and two High Efficiency Particulate Air (HEPA) capsule filters. A modified Microbiological Research Establishment type three-jet Collison nebulizer  with a precious fluid jar was used to generate a controlled delivery of aerosolized MPXV from an EMEM suspension. The Collison nebulizers are designed to generate aerosols having an approximate mean diameter of 1–2 μm. Aerosol concentration was determined by analysis of atmospheric samples drawn from a port on the side of the exposure chamber. The samples were collected by two methods in parallel: (1) into a glass impinger  filled with approximately 20 mL of media or (2) by impaction onto a 47 mm gelatin filter . A vacuum pump pulled the sample into the impinger or filter at 6.0 ± 0.3 L/min. The virus impacted on the gelatin filter was recovered by dissolving the filter in 10 mL of EMEM. The filter samples were enumerated by the plaque assay method to quantify the MPXV counts per milliliter. The gelatin filter enumeration results, along with the volume used to dissolve the filter, sampling rate, sample volume, and sampling duration, were used in the calculation of the aerosol concentration expressed as pfu/L of air .The Battelle large animal exposure system was used to expose animals individually, using a flow through design  were calculated from the Buxco XA software by the measured volumetric displacement of air caused by the movement of the thoracic cavity of an animal while it was in the plethysmograph. The data generated for each animal was used to determine the TATV, which along with the aerosol concentration was used in calculating the inhaled dose. The plethysmograph was connected to a pneumotach  that was attached to a differential pressure transducer . Pressure differential measurements from inhalations and exhalations were transmitted to Biosystems XA software that calculated and recorded respiratory function. Prior to animal exposures, the plethysmography system was calibrated to establish unit (baseline) and air volume displacements from 5 to 40 mL to simulate animal respiration. This calibration was performed to encompass the respiration volume range of the animal model for accurate TATV measurements.Clinical observations of each NHP were recorded twice daily for 21 days post-exposure. Body temperatures were collected once daily from two transponders (shoulder and hip) beginning two days prior to exposure through the end of study. Body weights were collected on Days 0, 2, 4, 6, 10, 14, 18, and 21 days post-exposure. Blood and buccal swabs for virus quantification by qPCR were collected on Days 0, 2, 4, 6, 10, 14, 18, and 21 post-virus challenge. The blood was collected in EDTA tubes, 200–400 μL. Lesion counts were obtained on Days 0, 2, 4, 6, 10, 14, 18, and 21 post-exposure. The weights, temperature, and lesion counts were tabulated on a Clinical Assessment form on Days 0, 2, 4, 6, 10, 14, 18, and 21 to aid in clinical monitoring.Necropsies were performed on all NHPs, and tissues collected for histology. Protocol-required tissue samples  were placed in 10% neutral buffered formalin, processed to approximately 5-micron H&E-stained slides, and examined microscopically by an ACVP board-certified pathologist. Gross and microscopic diagnoses were entered into the PATH/TOX SYSTEM  for data tabulation and analysis. Tissues from all NHPs were examined microscopically. Microscopic findings were graded semi-quantitatively according to the following scale, with the associated numerical score used to calculate average severity grades for each lesion by group and sex. Minimal (Grade 1) represented the least detectible lesion; mild (Grade 2) represented an easily discernible lesion; moderate (Grade 3) represented a change affecting a large area of the represented tissue; and marked (Grade 4) represented a lesion that approached maximal.Dinh) was calculated from the gelatin filter sample concentration (C), sampling parameters, total inhaled tidal volume (Vinh) and exposure duration (T) according to the equation below. The total number of MPXV captured during each exposure was the product of the gelatin filter concentration (Ci) and the volume the filter was dissolved in (C × V). The total number of PFUs was divided by the amount that was sampled through the filter during the exposure time (S × T). The aerosol concentration was (C × V) (S × T)−1. The inhaled dose was the product of the aerosol concentration multiplied by the TATV measured during plethysmography as detailed in Equation 1:The total inhaled dose C = Filter sample concentration (PFU/mL)Vi = Filter sample volume (mL)S = Filter sampling rate (L/min)T = Exposure time (min)Vinh = Total inhaled tidal volume (L), as determined by plethysmographyCa = Aerosol concentration (PFU/L)50 for the inhaled dose data was fitted to a logistic regression model to survival probability as a function of base-10 logarithm of the measured aerosol dose. Since the two lowest dose animals survived, and the four higher dose animals succumbed, a conventional logistic regression model such as Spearman-Karber would provide an indeterminate result. Thus, a logistic regression procedure that used a penalized maximum likelihood . Total nucleic acid was isolated from whole blood samples (100 μL) using the Specific B protocol on a NucliSens® easyMAG™ instrument . The homogenate was then poured through a 40 μM cell strainer, with the homogenizer and cell strainer rinsed with a total of 2 mL of NucliSens easyMAG Lysis Buffer. Buccal swabs were placed in a 1 mL solution consisting of 97% PBS, 2% FBS, and 1% Penicillin/Streptomycin. The entire recoverable volume of the solution was then added to 2 mL of NucliSens Lysis Buffer. Whole blood was collected into ETDA tubes, with 100 μL taken and added to 2 mL of NucliSens Lysis Buffer. Total nucleic acid was then isolated from these lysates using the NucliSens easyMAG. In brief, easyMAG Magnetic Silica is added to each sample after loading the lysate into sample vessels in the instrument. The Magnetic Silica then binds total nucleic acid during a 10 min incubation at room temperature, and the Magnetic Silica is then collected, and subsequently washed twice with NucliSens easyMAG Extraction Buffer 1, then twice with NucliSens easyMAG Extraction Buffer 2, and once with NucliSens easyMAG Extraction Buffer 3, with a final elution of total nucleic acid in 50 μL of NucliSens easyMAG Extraction Buffer 3.Total nucleic acid was isolated from whole blood samples, throat swabs and homogenized tissues using the Specific B protocol on a NucliSens® easyMAG™ instrument (BioMérieux) according to manufacturer's methods.7 to 2 copies/μL) reference standard curve with all samples to determine the concentration of gene copies in each sample. The final concentration for each sample was reported as copies per milliliter of original sample. The sequence of the primers, probe, and amplicon are as follows:Total purified nucleic acid was eluted in a final volume of 40 μL. Each nucleic acid sample was assayed by qRT-PCR for the MPXV N3R gene in duplicate on a 7900HT real-time PCR system . Each 25 μL reaction contained 5 μL of sample with the remaining volume consisting of TaqMan® Gene Expression Master Mix , sterile water, and a custom gene expression assay (Applied Biosystems) consisting of previously developed primers and a 3′-minor groove binding probe specific for a portion of the MPXV N3R gene  of 1.10 μm and ranged from 1.08–1.15 μm for each individual test, and the geometric standard deviation (GSD) was 1.61 μm with a range of 1.58–1.64 μm for each individual test. These results indicated that the aerosol distribution was relatively monodispersed and contained particles capable of reaching the deep lung in animal testing . The SF is the ratio of the concentration of agent in the aerosol (aerosol concentration) to the starting concentration used in the aerosol generator. As a consequence, the SF value is a unitless factor that represents the dilution that is to be expected during an aerosol experiment using the same bio-agent in the same aerosol system under the same conditions. This enables the assessment of the performance of the aerosol exposure process and whether it is stable (the same) for each individual test or exposure within a test day and across multiple test days.Following the aerosol characterization, the aerosol system was used to expose six cynomolgus macques to nebulized MPXV. The actual inhaled exposure dose was calculated for each animal by multiplying the actual inhaled air volume of the animal while in the aerosol chamber  against the aerosol chamber's virus concentration as measured by gelatin filtration. Table Table Virus burden in this study was measured by qPCR quantification of virus from tissues taken at necropsy and by qPCR quantification of virus in buccal swabs and whole blood taken every other day during the first 10 days after exposure. Low levels of virus were detected in buccal samples on Day 4 in one animal and in the blood of four animals. By Day 6 all animals had detectable levels in the blood and four out of six had detectable levels in buccal samples Table , suggestDose dependent virus shedding in saliva or viremia was not readily apparent, though the animal that received the highest exposure dose (C0404035) did appear to have higher blood and saliva virus titers than any others on any given day.As expected, the highest virus titers were found in lung tissues at necropsy Table  as this 4 pfu/animal group survived to the study end on Day 21 post-exposure. These animals had grossly evident skin pocks on multiple body sites but no internal gross lesions. All four monkeys exposed to higher concentrations of MPXV died or became moribund and were euthanized between study Days 7–10. Lung discoloration, bronchial lymph node enlargement, and/or skin pocks were evident in three of the four monkeys, while one lacked any gross lesions. All gross lesions noted were typical of monkeypox.Both monkeys in the targeted 3 × 10All monkeys that succumbed or became moribund during the study had microscopic lesions consistent with monkeypox. Lesions included surface (skin) and respiratory epithelial necrosis and reactive epithelial hyperplasia, with some epithelial cells in both sites containing eosinophilic to amphophilic intranuclear and/or intracytoplasmic inclusion bodies (virus inclusions). Fibrinosuppurative inflammation, edema, hemorrhage, and lymphoid necrosis were evident in the skin, tongue, lungs, and/or bronchial lymph nodes. Specifically, lung lesions had a prominent bronchial/peribronchial airway pattern (bronchopneumonia) indicative of the inhalation route of infection, as well as a slightly more subtle perivascular to interstitial pattern, consistent with a septicemic phase.50) and LD90 were estimated to be 77,900 pfu and 326,000 pfu, respectively. These results are similar to those reported in the recent literature , so 95% Fieller's confidence intervals for these percentiles are not bounded. The reason the 95% confidence intervals are not bounded is due to the small number of animals tested and because the two animals that received the lowest doses survived and the four higher doses animals died. Figure The median lethal dose  that received 9 × 105 pfu and 1 × 105 pfu survived longer than the two females (weight ≥3 kg). This question can only be resolved by conducting a well controlled age/weight/sex comparison study. To mitigate this potential affect, special consideration should be given to the size, age and sex of animals placed on future studies.The mortality data suggest that weight and/or NHP sex may be a contributing factor to this infection model. Two males, weight ≥8 kg, survived exposure with 3 × 10Virus in blood and buccal samples was detected as early as day 4 post-aerosol challenge and peak between days 6 and 10, which corroborates what has been observed by other investigators (Nalca et al., Multiple objectives were fulfilled from these tests. An aerosol exposure system was characterized using aerosolized MPXV, and it was shown that gelatin filters and impingers of type model 7541 generated equivalent enumeration results when sampling the MPXV aerosol. Following characterization of the aerosol exposure system, an NHP inhalation study determined a lethal exposure dose of MPXV, and an inhalation cynomolgus macaque model of MPXV infection was developed. Actual NHP exposures were performed with high precision relative to target dose, and the lesions and lethality were similar to the previously published data for the cynomolgus monkey (Zaucha et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective. The aim of this study was to evaluate the effectiveness of thiamine pyrophosphate against cisplatin-induced ototoxicity in guinea pigs. Materials and Methods. Healthy guinea pigs (n = 18) were randomly divided into three groups. Group 1 (n = 6) received an intraperitoneal injection of saline solution and cisplatin for 7 days, group 2 (n = 6) received an intraperitoneal injection of thiamine pyrophosphate and cisplatin for 7 days, and group 3 (n = 6) received only intraperitoneal injection of saline for 7 days. The animals in all groups were sacrificed under anesthesia, and their cochleas were harvested for morphological and biochemical observations. Results. In group 1, receiving only cisplatin, cochlear glutathione concentrations, superoxide dismutase, and glutathione peroxidase activities significantly decreased (P < 0.05) and malondialdehyde concentrations significantly increased (P < 0.05) compared to the control group. In group 2, receiving thiamine pyrophosphate and cisplatin, the concentrations of enzymes were near those of the control group. Microscopic examination showed that outer hair cells, spiral ganglion cells, and stria vascularis were preserved in group 2. Conclusion. Systemic administration of thiamine pyrophosphate yielded statistically significant protection to the cochlea of guinea pigs from cisplatin toxicity. Further experimental animal studies are essential to determine the appropriate indications of thiamine pyrophosphate before clinical use. Cisplatin is a mainstay chemotherapy drug in the treatment of a variety of solid tumors, notably testicular cancer. It is also used in the treatment of pediatric malignancies such as medulloblastoma and osteogenic sarcoma . CisplatInner ear toxicity is often a dose limiting side effect that hampers optimal cisplatin-based chemotherapy. It is normally manifested as a sensorineural hearing loss beginning in the high frequencies, successively progressing towards the speech frequency range . It is oCisplatin ototoxicity has several characteristics. In man it is mainly evident in the basal turn of the cochlea as degeneration of the outer hair cells (OHCs) and to some extent the inner hair cells (IHCs) and associated nerves . It has 1), and it is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. TPP plays a critical role in the carbohydrate and energy metabolism. In addition, TPP is involved in the α-oxidation of 3-methyl-branched and straight chain 2-hydroxy long chain fatty acids pathway functioning as coenzyme for peroxisomes. As a result TPP is a crucial cofactor for energy metabolism, antioxidation, and myelinization of nerve cells [Thiamine pyrophosphate (TPP) is the biologically active form of thiamine  was obtained from Liba Drug Company, Turkey. Thiamine pyrophosphate was obtained from SIGMA, Germany. All of the chemicals were of the highest quality commercially available.Eighteen healthy adult male albino guinea pigs weighing 1200–1500 g  were used in the current study. They had free access to water and food. The animals were kept under standard laboratory conditions, housed in a room at 20° ± 2°C temperature, and 12 h light/dark cycle. This study was performed with the approval from the Ataturk University Animal Care and Use Committee.n = 6) received an intraperitoneal (IP) injection of saline solution and cisplatin (5 mg/kg) for 7 days, group 2 (n = 6) received a n IP injection of TPP (25 mg/kg) and cisplatin (5 mg/kg) for 7 days, and group 3 (n = 6) received only IP injection of saline for 7 days (employed as control group).Guinea pigs were randomly divided into three groups and treated as follows: group 1  and their cochleas were harvested for morphological and biochemical observations. All surgical procedures were performed under a dissecting microscope with sterilized instruments.The level of endogenous antioxidant glutathione (GSH), the activities of antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and the concentration of malondialdehyde (MDA), the end product of lipid peroxidation, were determined enzymatically with the techniques explained by the literature –11.In order to avoid cell destruction by autolysis or bacteria and to preserve tissue morphology and composition, the cochleas were fixed in 10% neutral buffered formalin for 24 h at +4C° temperatures. Subsequently, decalcification was achieved by submerging the samples in 10% EDTA at room temperature for 7 days. The specimens were then washed with tap water and fixed again in 10% neutral buffered formalin for 24 h.μm of thickness were collected on glass slides and stained with haematoxylin and eosin staining. Sections were examined using a light microscope  and digital images were obtained by a digital camera (Olympus DP 71).Afterwards, the specimens were embedded in paraffin and then mounted in order to obtain mid-modiolar plane cuts. Sections of 5 P < 0.05.The SPSS statistical software, version 13.0 was used for the statistical analysis. Significance of the difference between the groups and subgroups were analyzed using the one way ANOVA test and Fisher's post hoc least significant differences (LSD). A difference was deemed to be significant at P < 0.05). Cochlear GSH concentrations, SOD and GSH-Px activities significantly decreased (P < 0.05) and MDA concentrations significantly increased (P < 0.05) in group 1, receiving only cisplatin, compared to the control group. In group 2, receiving TPP and cisplatin, the concentrations of GSH, MDA, and the activities of SOD and GSH-Px were near those of the control group. TPP restored the concentrations GSH and MDA and yielded statistically significant improvements in enzymatic activities of SOD and GSH-Px (P < 0.05) (There was a significant difference in level of GSH and MDA measurement and the activities of antioxidant enzymes GSH-Px and SOD among the groups ( < 0.05) .Samples obtained from the guinea pigs receiving only IP saline solution (group 3), revealed normal microarchitecture of the organ of Corti  . CisplatCisplatin have three important tissue targets in the cochlea, including organ of Corti, spiral ganglion cells, and lateral wall . In guinea pigs that received consecutive cisplatin applications, destruction of outer hair cells and myelin sheath detachment of spiral ganglion cells were observed . FurtherNumerous studies suggest that cisplatin induced hearing loss is mostly related with generation of ROS, initiating cascade oxidative mechanisms. Despite the presence of endogenous antioxidant molecules, including glutathione and the antioxidant enzymes, cisplatin induced oxidative stress can overwhelm these intrinsic defense mechanisms. Consequently, exogenous administrations of antioxidants have been the primary focus in the treatment of cisplatin induced ototoxicity .At present, the only way to prevent cisplatin-induced ototoxicity is a limitation of the total dose per cycle, the cumulative dose, and the dose intensity , 17. Obv1), upon entry into cells, thiamine is quickly converted to TPP that is the active substance. However, recent studies indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated [TPP is the biologically active form of thiamine (vitamin Borylated , 22. ThaIn many forms of ototoxicity, pharmacological activation of intrinsic defense mechanisms could be helpful. Cisplatin induced ototoxicity is a special issue in that the ototoxic insult is predictable. It should be possible to administer protective agents at precisely timed intervals before the insult. The results of this present study suggest that TPP is beneficial in reduction of experimental cisplatin ototoxicity in guinea pigs, and it may be a potential candidate drug in human beings.We have demonstrated the efficacy of systemic administration of thiamine pyrophosphate in the prevention of cisplatin induced ototoxicity using a guinea pig model. Further experimental animal studies are essential to determine the appropriate indications and dosages of TPP before clinical use.


Obesity and diabetes are increasing in prevalence worldwide. Despite excessive dietary consumption, obese individuals have high rates of micronutrient deficiencies. Deficiencies of specific vitamins and minerals that play important roles in glucose metabolism and insulin signaling pathways may contribute to the development of diabetes in the obese population. This paper reviews the current evidence supporting this hypothesis. Obesity is highly prevalent both in the United States and worldwide and is projected to surpass tobacco use as the most economically important modifiable risk factor in public health and disease –3. Multiβ-cell function and in the insulin signaling cascade suggests that deficiency in these micronutrients may play a role in the development of type 2 diabetes.The importance of certain micronutrients as cofactors in glucose metabolic pathways, pancreatic β-cell dysfunction, and genetic and behavioral factors all contribute to the development of diabetes in obese individuals [The risk of type 2 diabetes is increased 4-fold in obese patients . The cauividuals . Specifi High rates of vitamin D insufficiency and frank deficiency have been reported in obese individuals and in diabetics. The prevalence of vitamin D insufficiency (defined as <30 mg/dL) in obese individuals ranges from 80–90% , 9. Whilβ-cells, the detection of 1-α-hydroxylase activity, and insulin gene transcription responsiveness to vitamin D in pancreatic β-cells [β-cell dysfunction is noted, which is restored with vitamin D supplementation [ Preclinical evidence for the role of vitamin D in insulin secretion and function includes the presence of vitamin D receptors in human pancreatic  β-cells –16. In ventation –19. Manyentation –25. Seasentation , 27. Recent reviews and metaanalyses of clinical trials suggest a potential effect of vitamin D supplementation on the development of type 2 diabetes in high-risk individuals , 16, 28.The use of vitamin D in patients already diagnosed with type 2 diabetes has also been investigated. Of four recently published randomized trials, two demonstrate improvements in glycemic control –36. In oIn one of the trials that demonstrated negligible effect of vitamin D supplementation in type 2 diabetics, patients given single dose of 300,000 i.u. of cholecalciferol found no change in fasting glucose, fructosamine, or insulin sensitivity despite normalization of plasma 25-hydroxyvitamin D levels . HoweverThe other negative study was a randomized trial with 6 weeks of followup in which 32 diabetic patients were given 40,000 i.u. of cholecalciferol or placebo every two weeks and showed no difference in glycosylated hemoglobin, fasting, glucose or insulin sensitivity . Subject The use of vitamin D supplementation specifically for improvements in insulin sensitivity remains a controversial issue. There are presently ten ongoing clinical trials evaluating the use of vitamin D supplementation in type 2 diabetic patients and patients with impaired glucose tolerance, which may provide further guidance on this issue. Given the high rates of vitamin D insufficiency in the obese population and the observed benefits, clinicians may consider vitamin D supplementation in this population. Chromium was recognized as an essential trace metal required in the insulin signal cascade through animal dietary experiments that were first performed in the 1950s , 38. MolChromium deficiency in humans has been identified in severely malnourished patients who demonstrate severe insulin resistance, hyperglycemia, hypertriglyceridemia, and painful neuropathy. In published case reports, these symptoms and signs completely and rapidly resolve after the administration of chromium –44. As aThe main dietary sources of chromium include yeast, meats, and wheat germ. The use of stainless steel pots and utensils increases the chromium content of food as traces of chromium are liberated from the steel during preparation. Body stores of chromium are intracellular, mainly in the liver. Individuals with type 2 diabetes have 20–40% lower blood chromium levels and 40–50% lower chromium levels measured in scalp hair compared to healthy controls –49. ChroA number of clinical trials have investigated the role of oral chromium supplementation in patients with type 2 diabetes, insulin resistance, and the metabolic syndrome , 51. In As with many supplements, the use of chromium supplementation in diabetic patients or in patients at risk for diabetes is controversial. The results of clinical trials that administer high doses of high bioavailability chromium suggest that supplementation with chromium may improve insulin resistance and glycemic control in diabetes . Recentl Biotin is a water soluble vitamin that serves as a cofactor for carboxylase enzymes in fatty acid synthetic pathways, the citric acid cycle, and aminoacid metabolism . In addiβ-cell function [Animal studies demonstrate the development of insulin resistance in biotin deficient rats that resolves with biotin replacement . At leasfunction , 66.To date, only a few human trials evaluating the efficacy of biotin supplementation on glycemic control have been conducted. In one longitudinal study, diabetic patients treated for 28 days with 15 mg biotin supplements had improvements in fasting glucose and insulin levels , 67. Two With the promising results of these early studies, the potential benefits of biotin supplementation may be considered in diabetic and obese patients.α-ketoglutarate dehydrogenase, and α-keto acid decarboxylase [ Thiamine is an essential micronutrient that acts as a cofactor for several key enzymes in glucose and aminoacid metabolism including transketolase, pyruvate dehydrogenase, boxylase . The latboxylase . Thiaminboxylase   trial, in which 3654 diabetic patients were randomized to receive 400 i.u. of  placebo . After 4 placebo –92. Alth placebo , 94 a re placebo .  Vitamin C or ascorbic acid is a water soluble antioxidant vitamin that does not have significant body stores. Limited intake of fruits and vegetables can lead to rapid depletion . Plasma In a population study of 232,007 older adults, vitamin C supplementation was associated with mildly lower rates of type 2 diabetes in a dose responsive manner. Individuals that took 500 mg daily had a 9% reduction in prevalence of diabetes . Data frFew clinical trials have examined the use of vitamin C supplementation in type 2 diabetic patients. One small randomized trial in 20 diabetic subjects demonstrated that 1000 mg daily oral vitamin C supplementation improved glucose disposal rates after 4 months . A seconFurther studies in diabetic patients and in those at risk for developing diabetes may help to elucidate the role of vitamin C supplementation in this population. The high rate of vitamin C deficiency in obese individuals suggests that supplementation may be beneficial. Increasing dietary intake of fruits and vegetables can also address this deficiency and is currently recommended as part of a lifestyle intervention for the prevention and treatment of type 2 diabetes . As with nearly all biochemical processes, glucose metabolism and insulin signaling require cofactors and vitamins that are essential in the diet. Deficiencies in any of these micronutrients have potential to impair glucose metabolism and cause insulin resistance. Clinical evidence supporting this hypothesis regarding the metabolic effects of specific deficiencies including vitamin D, chromium, biotin, thiamine and vitamin C is mounting. Unlike vitamin E, which has little to no proven clinical effect when given as a supplement, these vitamins are known to be deficient at relatively high rates in obese individuals and in diabetic patients. Clinicians should consider addressing possible deficiencies of these micronutrients when advising obese patients who are at risk for the development of type 2 diabetes. The medical care plan for obesity should include lifestyle changes, healthy food choices with high-nutrient content foods as part of a balanced approach for the prevention of the development of type 2 diabetes. Use of specific vitamin supplements may adopted into this rational practice.


Monolayer cultures of cells that migrated from individual granulomas were established in the proposed culture settings for mouse spleen and lung granulomas induced by in vivo exposure to BCG vaccine. The cellular composition of individual granulomas was analyzed. The expression of the leukocyte surface markers such as phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and the expression of the costimulatory molecules CD80, CD83, and CD86 were tested as well as the production of proinflammatory cytokines (IFNγ and IL-1α) and growth factors (GM-CSF and FGFb) for cells of individual granulomas. The colocalization of the phagocytic receptors and costimulatory molecules in the surface microdomains of granuloma cells (with and without acid-fast BCG-mycobacteria) has also been detected. It was found that some part of cytokine macrophage producers have carried acid-fast mycobacteria. Detected modulation in dynamics of production of pro-inflammatory cytokines, growth factors, and leukocyte surface markers by granuloma cells has indicated continued processes of activation and deactivation of granuloma inflammation cells during the latent tuberculous infection progress in mice.The new  Mycobacterium tuberculosis as the causative agent, accounts for about 2 million deaths annually and is one of the leading causes of deaths from infectious disease caused by a single agent. According to WHO reports, one-third of the world's human population is infected with M. tuberculosis and each infected with this bacterium has a 5–10% risk of developing active tuberculosis, which amounts to 8-9 million new cases annually. Intracellular presence of mycobacteria in specific inflammatory lesions called granulomas is one of the known features of the tuberculosis. Granulomas are the aggregates of the immune cells, predominantly macrophages, containing mycobacteria [Tuberculosis, with the intracellular parasite bacteria . Granulobacteria . Macrophbacteria –11.γ is the most important reaction controller for both innate and adaptive immune responses [γ, produced mainly by T cells, supports inflammatory response progress, which also includes granuloma formation [γ activates macrophages leading to the increase in their phagocytal and microbicidal functions. It also promotes production of the proinflammatory cytokines (IL-1 and TNFα) and growth factors (GM-CSF and FGFb) that are involved in cell differentiation and aging in the inflammatory tissue. The IFNγ is also the most important transmitter in the elaboration of adaptive Th1-dependent response manifested by the increase of the expression of MHC class I and II proteins and costimulatory molecules CD80, CD83, and CD86 on the surface of antigen-presenting cells [γ, absorb mycobacteria, opsonized by specific antibodies and complement, with the phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and destroy them by means of producing antimicrobial effectors such as oxygen radicals and nitric oxide [γ. As observed in IFNγ-deficient mice, subjects with defects in IFNγ are profoundly susceptible to mycobacterial infections [α and secreted IL-1β, are also considered to be basic transmitters of the inflammatory response. They have a wide range of biological potency, including the increase in immune cell resistance to bacterial infections via cytokine synthesis and growth-stimulating factor induction. GM-CSF, produced by activated macrophages, fibroblasts, and T-lymphocytes, is a hematopoietic factor that stimulates the development of neutrophils and macrophages and enhances their functional antibacterial activity, as well as takes part in proinflammatory cytokines synthesis induction [Cytokines play the most important role in controlling immune system function as a response to a contagion. It was shown that in tuberculosis infection, IFNesponses , 8, 12. ormation , 12. Theic oxide , 7. It wfections , 7. Bothnduction . FGFb, anduction .Danio rerio) juveniles and mice have been recently developed to analyze the individual granulomas [M. marinum allowed the analysis of the initial stages of the formation of macrophage granulomas and their responsiveness to antituberculous therapy [M. bovis allowed to describe the dynamic behavior of macrophages and lymphocytes especially their interaction within granulomas critical for the induction and regulation of antituberculous immunity [During the last years the expression of surface markers and cytokines in bulk population of tuberculosis granuloma cells, obtained mostly from body organs of several mice and then treated with collagenase to destroy extracellular structures, was analyzed using flow cytometry method –23. Howeanulomas –28. Stud therapy –27. Realimmunity . Consequimmunity . New sysex vivo culture system after the initial granulomas were induced in mice by infection with BCG vaccine in vivo. As a result, monolayer cultures of cells migrated from individual granulomas were established and maintained in the proposed culture settings. The dynamics of granuloma formation and functional contributions of granuloma cells of different types as well as with the analysis of surface markers, cytokines, and growth factors to promoting the growth of granuloma inflammation in mice with latent BCG infection has been studied using these granuloma cell cultures. In the present work the granuloma cells were studied using the new BALB/c mice aged 2 months were obtained from the Animal Breeding Facility of the Institute of Cytology and Genetics, Siberian Branch (SB) of Russian Academy of Sciences (RAS), Novosibirsk, Russia, and kept under standard vivarium conditions in accordance with all the known guidelines for laboratory animal care. RPMI 1640, phosphate buffer saline (PBS), fetal bovine serum (FBS), glutamine, and gentamicin were obtained from BioloT . All other chemicals were obtained from Sigma-Aldrich . APC-labeled antibody against mouse CD11b was obtained from eBioscience . All other primary specific antibodies, secondary specific antibodies labeled by biotin, and horseradish peroxidase conjugated streptavidin were purchased from BD Pharmingen . FITC-labeled antibodies against rat or mouse immunoglobulins (Ig) were obtained from Abcam . Tissue-Tek O.C.T. Compound was obtained from Sakura (the Netherlands). VECTASHIELD Mounting Medium with DAPI  was given as a gift by Vector Laboratories . M. bovis  at a dose of 0.5 mg per mouse, which amounted to 3 × 106 viable BCG-mycobacteria in 0.9% NaCl solution. Twenty-three mice were infected via tail vein injections with 100 μL of the suspension per mouse and two mice were infected intraperitoneally with 200 μL of the suspension per mouse. Mice were infected with the vaccine prepared from an attenuated live strain of μm slides on Microtome cryostat HM 505N  at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, SB RAS, (Novosibirsk). Sections were air-dried on slides and stained with azure-eosin and methylene blue.The spleens and lungs of mice were removed and cut into pieces in RPMI 1640 medium. Parts of tissues from some mice were collected and fixed with 4% formaldehyde solution in PBS (pH 7.4) for 90 min at room temperature. After fixation, the tissues were washed with PBS, incubated with 30% sucrose in PBS (pH 7.4) for 20 hours at +4°C, frozen in Tissue-Tek O.C.T. Compound at −25°C, and sectioned at 16 μg/mL gentamicin into small pieces. For homogenization the dissected lungs were further disrupted by gently pushing the tissue through a metal screen with pores of 1 mm diameter. Granulomas were isolated from the organ homogenates by centrifugation at 150 ×g and washed three times in RPMI 1640 medium with 50 μg/mL gentamicin. Granuloma pellets in a complete growth medium containing 10% FBS, 2 mM glutamine, and 50 μg/mL gentamicin were placed at low-medium density to 24-well plates  with cover glasses in the bottom and cultured in 0.5 mL medium for several days at +37°C in atmosphere containing 5% CO2. FBS inactivated by heating for 30 min at +56°C was used. The isolation of granulomas from the spleens and lungs of mice of two groups after one and two months lasting infection was performed as described by Sacco and the colleagues  without ex vivo culture, granuloma cells on cover glasses were fixed with 4% formaldehyde solution in PBS (pH 7.4) for 10 min at room temperature. Before staining, the preparations were kept in 0.4% formaldehyde solution in PBS (pH 7.4) at +4°C. To visualize acid-fast bacteria, some of the preparations with granuloma cells were washed with PBS and stained by Ziehl-Neelsen method according to standard protocols. The cells were further counterstained with 1% methylene blue. The other preparations were stained using antibodies raised against cell surface markers. The preparations of cultured and fixed, as granuloma cells, peritoneal macrophages of normal BALB/c mice without BCG infection were used as the control groups. The control fibroblasts were obtained by spontaneous cell migration from fragments of the lung tissue or the splenic capsule from intact BALB/c mice in the wells of 24-well plates with cover glasses on the bottom in minimal amount of a complete growth medium at +37°C in atmosphere containing 5% CO2. After removing tissue fragments the migrated fibroblasts on cover glasses were further cultured for optimal density in 0.5 mL medium for several weeks. Several markers were stained for using the preparations of peritoneal macrophages obtained from mouse 25 after two months of intraperitoneal BCG infection and cultured as granuloma cells. After 2–5 days of α were diluted 1 : 10 and 1 : 50, respectively. The rat antibodies to IFNγ, GM-CSF, CD11b, CD14, and CD16/CD32 were diluted in 1 : 100, 1 : 25, 1 : 250, 1 : 50, and 1 : 50, respectively. The mouse primary antibodies to FGFb were diluted 1 : 100. The goat secondary polyclonal antibodies against rat Ig were diluted 1 : 25; the mouse secondary polyclonal antibodies against hamster Ig were diluted 1 : 50. Specific staining was visualized using horseradish peroxidase conjugated streptavidin in diaminobenzidine solution containing 0.05% H2O2. The cells were further counterstained with 1% methyl green. Fluorescent visualization of the proteins was enabled using FITC-labeled antibodies against rat or mouse Ig diluted 1 : 400. Some of the cell preparations were stained with antibodies against CD80, CD83, or CD86 labeled by PerCP-Cy5.5, APC, or PE-Cy7, respectively, diluted 1 : 100. Immunofluorescent staining of cells was analyzed using VECTASHIELD Mounting Medium with DAPI. After recording of the confocal images of the cells, the preparations were washed of VECTASHIELD Mounting Medium in PBS during 20 minutes and restained for acid-fast mycobacteria using Ziehl-Neelsen stain. Cell preparations were washed with PBS, treated within 0.5–2 minutes in 0.3% Triton-X100 solution (only for cytokines staining), blocked in PBS solution containing 2% BSA, and finally incubated first with primary specific antibodies and then with secondary specific antibodies labeled by biotin or fluorochromes. The hamster primary antibodies to CD11c and IL-1plus microscope (Zeiss) and objectives with various magnifications (Zeiss), photographed using an AxioCam HRc camera (Zeiss); the images were analyzed using the AxioVigion 4.7 microscopy software (Zeiss). Proteins stained with fluorescent dyes were examined under an LSM 510 or LSM 780 (Zeiss) confocal microscopes using the LSM Image Browser and ZEN 2010 software (Zeiss). The histological and cytological preparations were examined at the Shared Center for Microscopic Analysis of Biological Objects of the Institute of Cytology and Genetics, SB RAS, using an Axioscop 2 t-test.Statistical data processing was performed using MS Excel 2007 (Microsoft). Differences were tested for significance using Student's Granulomas were isolated from the spleens and lungs  of mice after one month (/1) and two months (/2) of being infected with the BCG vaccine and seeded into culture plates. Granulomas obtained at one month and two months after infection will be denoted as Gran/1 granulomas and Gran/2 granulomas, respectively, throughout. Gran/1 granulomas were from mice 1 ÷ 5; Gran/2 granulomas were from mice 1 ÷ 16 and 21 ÷ 25. Mice 1S/1 and 25S/2 were infected intraperitoneally; the others were infected via tail vein injections. By the time of isolation, none of the mice had been observed to have acute tuberculous disease. Therefore the granulomas were isolated from mice with latent chronic BCG infection. All granulomas examined in lungs and spleens of BCG-infected mice on histological sections were solid Figures . So as iex vivo at low-medium density using heat-inactivated FBS without complement proteins for a varying number of days, from 2 to 5 on most occasions. Monolayer cultures of cells that had migrated from individual granulomas were established in the proposed culture settings  on histological sections of tissues. The acid-fast mycobacteria were detected in granuloma cells isolated from different organs of all examined mice in ex vivo culture  than Gran/1 granulomas  (P < 0.05). Interestingly, granulomas in zebrafish juveniles had an average length of 85–105 μm on days 3–5 after being infected with M. marinum [Granuloma size in the spleens of mice of both groups, infected for one and for two months with BCG, was measured after seeding  marinum . The cellular composition was inferred for each granuloma individually. The granulomas were largely composed of macrophages varying in population size Figures . All theBesides macrophages, cells of nine more types were observed in the granulomas from mice with latent BCG infection: dendritic cells, lymphocytes, fibroblasts, neutrophils, eosinophils, megakaryocytes, thrombocytes, erythrocytes, and multinucleate Langhans giant cells. The population sizes of cells of other types were much lower and still varying therefore they were analyzed as the percentage out of the macrophage population in granulomas in The few granulomas isolated from the lungs of different mice were found to have the same cellular composition as the granulomas isolated from the spleens. In the lung granulomas, macrophages were the major cell type, the dendritic cell population was the same as in the spleen granulomas, and fibroblasts showed an occasional occurrence. The main difference between mouse lung and spleen granulomas was that the former had extremely small lymphocyte populations.The average dendritic cell population in the mouse spleen granulomas remained constant during latent BCG infection and was about 10% of the macrophage population whether in Gran/1 or Gran/2 granulomas see . DendritP < 0.05, see P < 0.01). The percentage of fibroblasts in the examined granulomas did not exceed 15% of the macrophage populations, but granulomas with the fibroblast populations made up more than 40% of the observed macrophage populations  had changed from mouse to mouse as well as the number of these cells within granulomas, the range being 5–13% of within-granuloma macrophage populations. Noteworthy, the neutrophils remained viable in all granuloma cell cultures Figures ; even thex vivo culture, these cells continued to form by fusion of the membranes of Langhans giant cells with adjacent macrophages (but not with dendritic cells) and by admission of new macrophage nuclei to the nuclear aggregates . Occasionally, thrombocytes, formed due to cytoplasmic fragmentation, were observed in these cells , and megakaryocytes in leukocyte infiltrates, obtained from the spleen of the mouse 6S/2, while fibroblasts and eosinophils were not observed at all see .ex vivo suggests that none of those cells was apoptotic or necrotic. Few lymphocytes had segmented nuclei, while the integrity of the cell membranes was not compromised. Our observation of the morphology of the macrophages, dendritic cells, segmented leukocytes, fibroblasts, megakaryocytes, and Langhans giant cells in the mouse granulomas cultured μm. CD14, the receptor for lipopolysaccharide in bacterial cell walls, occurred at an increased density on the membranes of the peritoneal macrophages in the control mice and macrophages and dendritic cells in the granulomas  and leukocyte surface markers specific for dendritic cells  were found on both cells types within the granulomas examined Figures . Examineanulomas . The celanulomas . Few graanulomas . Both phanulomas . NeitherP < 0.001). The average number of the macrophages that contained the phagocytic receptors CD11c and CD16/CD32 was largely found in Gran/2 granulomas, while dendritic cells stained for leukocyte surface marker CD11c were largely found in Gran/1 granulomas (Granuloma cells (macrophages and dendritic cells) with expression of various surface antigens were analyzed in each granuloma separately, and the figures were averaged over the mice and then combined for Gran/1 and Gran/2 granulomas Figures . Many maanulomas . No diffanulomas . All macγ, IL-1, and GM-CSF are inflammatory mediators that activate and differentiate immune cells increasing their microbicidal potential in response to infection by intracellular pathogen thus playing an important role in initiating, regulating, and control of inflammation [γ, cell-associated IL-1α, GM-CSF, and FGFb detected significant production activation for examined cytokines in granuloma macrophages and leukocyte infiltrates of mice after one and two months of being infected with BCG comparing to the peritoneal macrophages from the control group of untreated mice , though their amount in Gran/2 granulomas did not exceed 25% at the average , size, and the number of cells in them both within each mouse and between the mice. Interestingly, diffuse leukocyte infiltrates containing increased numbers of dendritic cells and neutrophils, which are the hallmarks of acute rather than chronic tuberculous infection, were observed even in the spleen of mouse 6S/2 infected with BCG for two months, where the number of well-formed granulomas was the lowest. It is possible that either the initial stages of granuloma formation in the mouse spleen or, on the contrary, developing active tuberculosis after short-term chronic tuberculosis were observed. Every hypothesis leads to additional investigations. As it is well known, most of BALB/c mice are resistant to developing active tuberculosis [The approach that we used for culturing mouse granulomas rculosis , but botex vivo culturing conditions involving heat-inactivated FBS without complement proteins. It can therefore be hypothesized that, under certain conditions within the organism, these cells can also actively travel across the organs and tissues and disseminate mycobacteria to them. It is possible that the difference in the number of granulomas isolated from the spleens of different mice is indicative of the different abilities of cells in migrating from granulomas in some mice and, consequently, in forming new granulomas within the same organ. It is also possible that the different sizes of these aggregates could be due to the different times of granuloma formation: larger granulomas must be older, while the smaller formed later from cells that had migrated from the older. However, these possibilities can still only be hypothesized upon due the insufficient knowledge of the processes underlying the emergence, development, optimal size, cellular composition of granulomas, and cell rotation, which are required, on the one hand, for restraint of infection and survival of the host and, on the other hand, for the replication and dissemination of the pathogen—such that the delicate trade-off is reached in the latent tuberculous infection. The question as to whether the granuloma structure remains closed at all times during latent infection or otherwise is one of the most critical issues in the matter of understanding mycobacterial dissemination to organs and tissues. It has recently been demonstrated that macrophages were highly mobile at the initial stage of tuberculous infection in zebrafish juveniles , 33, whiin vitro models have ever revealed megakaryocytes in granulomas [Our studies confirm that macrophages did play a key role in the pathogenesis of latent tuberculous infection in the mouse spleen and lung –12, 31. anulomas , 15, 21.anulomas , while iThe fact that dendritic cells were present in all the mouse granulomas suggests that this cell type is important for the development of latent BCG infection. The dendritic cell population is estimated as not higher than 0.1% of blood mononuclear cells in animals . HoweverPhagocytic and costimulatory markers were found on the membranes of macrophages and dendritic cells in the mouse granulomas but not on the control peritoneal macrophages (with the exception of the receptor CD14). The active status of cells in the mouse granulomas was indicated by both higher expression of the leukocyte surface markers and a microdomain organization of these receptors on the granuloma cells and their colocalization with each other in most microdomains on granuloma cell membranes. The question as to whether the observed microdomains are rafts and, therefore, places for transducing cellular signals to the effectors below, which modulate the behavior of mouse granuloma cells in latent BCG infection, has yet to be answered. As is known, to be able to ingest bacteria, the host's cells should have phagocytic receptors clustered on their membranes . Therefoγ, IL-1α, and GM-CSF in macrophages during the progress of  latent tuberculous infection in mice. If more than a half of leukocyte infiltrate macrophages of the mouse 6S/2 were producing IFNγ, then there were less such cells detected in Gran/1 granulomas, but the amount of macrophages producing IFNγ significantly increased in Gran/2 granulomas. Synthesis of IL-1α and GM-CSF was detected mostly in macrophages of Gran/2 granulomas, and it shows that there was also ongoing activation of macrophages during the progress of the granulomatous inflammation in mice with latent tuberculous infection. It is probably the granuloma cells producing GM-CSF and FGFb growth factors that promoted their survival in both compact structures of granulomas and ex vivo culture. Inverse dynamics of producing IFNγ was studied in granuloma lymphocytes: almost all of the lymphocytes of Gran/1 granulomas contained IFNγ in cytoplasm; however, there were very few lymphocytes producing IFNγ detected in Gran/2 granulomas. IFNγ, released by T lymphocytes, is considered to activate macrophages and dendritic cells in human and animal granulomas for struggling with tuberculous infection [γ production changed, that were observed during the progress of latent tuberculous infection in mice. However the reason of these changes is yet to be studied. Generally the investigation of synthesis of cytokines and growth factors in the granuloma cells, with or without BCG-mycobacteria, during the granulomatous inflammation in mice is necessary. Studying of cytokine production in granuloma cells showed modulation of expression of IFNnfection , 8, 12. γ and other cytokines, phagocytic and costimulatory markers in granuloma macrophages and dendritic cells, with or without acid-fast BCG-mycobacteria, were detected. Therefore, some of the activated macrophages and maturated dendritic cells of mouse granulomas did not manage to destroy intracellular BCG-mycobacteria. This observation disagrees on the issue that activated macrophages and maturated inflammatory dendritic cells must successfully destroy intracellular bacilli [ It is worth noting that the production of IFN bacilli , 35, 36.ex vivo model system developed we studied the impact of individual granuloma cells to growth and control of the granulomatous inflammatory lesions in mice with latenttuberculous infection. Granulomas from the mouse spleens and lungs were largely composed of macrophages; however, they also contained dendritic cells, lymphocytes, and occasionally fibroblasts, neutrophils, eosinophils, megakaryocytes, and multinucleate Langhans giant cells formed by fused macrophages. It has been demonstrated that all polymorphonuclear leukocytes, many macrophages, dendritic cells, lymphocytes, megakaryocytes, and Langhans giant cells of mouse granulomas, with or without BCG-mycobacteria, substantially expressed the phagocytic receptors, costimulatory molecules, and proinflammatory cytokines on high level. Also the modulation of the activation of granuloma cells  during the progress of the granulomatous inflammation in mice with latent tuberculous infection was detected. Therefore the suggested new ex vivo model of granuloma cell cultures allowed not only to evaluate the input of cells of different type to the growth of inflammatory granulomas by its functional means but also to start studying the intercourse of mycobacteria, the intracellular pathogen, and host cells during latent stage of tuberculosis infection in mice. The reported study helps to elucidate why vaccines and therapeutic treatment seldom rid the organism of tuberculous infection.With the


For today’s highly precise hematology analyzers, the following control rules are strongly advised: 1s3.5, 1s4 and 1s4.5 rules . In order for the hematology laboratory to totally embrace expanded QC limits, manufacturers must make available their instruments’ usual and poorer (e.g. the 15P performance) imprecision’s. Users of hematology analyzers that require more sensitive but less specific rules to prevent the reporting of clinically erroneous data are advised to acquire more precise (and thus more dependable) instrumentation.Diverse approaches have been used to assure the analytical quality of automated hematology; as such, there is great variation in their error detection capabilities. We summarize the intralaboratory performance of a cohort of Sysmex XE-2100’s running e-Check hematology quality control (QC). The imprecisions of a median performing (50th percentile imprecision) and more imprecise [15th percentile (15P) imprecision] Sysmex XE-2100 are compared with measures of total allowable error  to obtain multiples of the usual imprecision that must be detected to prevent the hematology analyzer from producing medically unacceptable results. The resultant large multiples of the usual imprecision ( Diverse approaches have been used to assure the analytical quality of automated hematology analyzers. These approaches encompass the re-analysis of retained patient specimens  averaginvs. the size of error. Westgard has provided power function curves for control rules based on the analysis of reference sample materials   . Cembrowaterial) . These paterial) .While virtually all laboratories in developed countries analyze commercial control materials for hematology QC, there is significant variation in the setup and interpretation of the hematology QC. The control limits are variable with some laboratories using the limits supplied by the manufacturer, and others using control limits that are derived statistically from preliminary analysis of the new QC material. The frequency of control material analysis varies. Some laboratories analyze controls every 8 or 12 h. Other laboratories ‘bracket’ groups of patient specimens with QC specimens or retained, previously analyzed specimens. There is also significant variation in the control rules and control procedures (combinations of QC rules applied in a defined manner) used to interpret the QC data. A 1994 College of American Pathologists Q-Probe surveyed the QC practices in 505 primarily US health care institutions (39% teaching and 61% nonteaching) . OverallIt is incongruous that the quality of today’s multichannel hematology analyzers is generally undisputed, yet the practice of hematology QC is heterogeneous and seemingly arbitrary. It may be that these analyzers are robust and usually produce clinically acceptable data and that many of today’s QC procedures are nonspecific and lead to nonproductive practices. In this study, we evaluate the stability of e-Check hematology QC product as analyzed by a cohort of laboratories using a representative robust hematology analyzer, the Sysmex XE-2100  limits fa) (MAE:CVa) dictates the QC rule that should be used to detect analytically important error. A high ratio (exceeding 4–5) indicates a very tightly controlled analytical process. For such high ratios, QC rules such as the 1s3.5, 1s4 and 1s4.5 are adequate. For MAE:CVa values of 3–4, tighter control limits are required to detect analytically significant error. A control procedure combining the 1s3 and the 2s2 control rules will generally suffice for these intermediate ratio tests.The ratio of MAE to the analytical imprecision . It provides a 14-parameter hemogram, a 5-part leukocyte differential, reticulocyte analysis including immature reticulocyte fraction, and a nucleated red blood cell (NRBC) count. Differential parameters, reticulocyte analysis, and NRBC counts are determined using flow cytometry, a semi-conductor laser and fluorescent dyes . The SysThe Sysmex XE-2100 uses a three level control product, Sysmex e-Check, for process QC. This material consists of a stabilized whole blood mixture of human erythrocytes, human and simulated leukocytes and platelets (PLT). If promptly refrigerated after each use, the material has a 73-day closed vial stability and a 7-day open vial stability. pdf format  for the periods of July 17, 2002 to August 15, 2002 (period 1) and August 16, 2002 to September 20, 2002 (period 2). These summaries were converted into text files and the text data extracted into Microsoft Access, Crystal Reports and Microsoft Excel to provide data summaries and graphs.The QC data are transmitted regularly from the Sysmex XE-2100 to the Sysmex Insight Program. This program provides calculations of the individual laboratory’s mean and SD for each control and analyte as well as corresponding interlaboratory statistics. These calculations are continuously available via the World Wide Web. Group summary reports are also produced at the end of each 30-day period. We obtained two 30-day summaries of submitted data for Sysmex e-Check lot number 2197 from 121 different laboratories in We tabulated the individual Sysmex Insight intralaboratory CV for each analyte and determined the following CV: 10P, 15P, 50P, 85P and the 90P, 95P and the 99P with the highest percentiles corresponding to the most precise performance. Graphs were constructed showing the 10–99P CV for the analytes shown in a ratios for both the 15P and 50P CV. We also calculated the error magnitude (measured in number of SD's) required in the results before an error would exceed the manufacturer’s limits. Finally, the differences between the MAE:CVa ratios and the QC error magnitudes were calculated  did not provide period 2 control data. On average, 60 controls were analyzed at each control level.In period 2, reports were available for 153 analyzers from 118 laboratories; 32 of which used multiple analyzers. Nine analyzers ran QC both in the ‘closed’ and ‘open’ sampling mode, for a total of 162 sets of data. One hundred and forty nine sets of data were obtained in the ‘closed’ mode and the remainder was run ‘open’.On average, 70 controls were analyzed at each control level. In a sample of 50 hospital laboratories that maintained 24-h services, 46% ran QC three times per day, 34% ran two times per day, 14% four times a day and 6% ran QC only once a day.P< 0.01). a ratio >4 except for mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV). The period 1 and period 2 CV are somewhat larger than those demonstrated in an early XE-2100 evaluation . Publisha ratios exceed 5 for all analytes for the 15P (below average) analyzers  and absolute basophil/monocyte counts.When using the CLIA limits, the MAE:CVnalyzers . When thnalyzers . When usl limits . HoweverP< 0.05 physiologic limit is exceeded for leukocyte, neutrophil, eosinophil and basophil counts, Hgb, PLT and RBC. With the use of P< 0.01 physiologic limits, this list of analytes would also include mean platelet volume (MPV) and Hct. Of these tests, Hgb, PLT, leukocyte count and neutrophil number, are probably the most clinically useful and most commonly ordered tests. With reference to those tests for which both physiologic limits are narrower than the QC limits, three of the tests, MCV, MCH and MCHC are calculated by the Sysmex. We have found that the investigation of deviations in calculated parameters is not productive as these deviations usually arise from simultaneous errors in two or more of the constituent measurements.Using the CLIA limits, the manufacturer QC limits are narrower than the MAE for the 15P analyzer  as well 9) is similar to the physiologic limit while the QC limits for monocyte number ( ± 0.4 × 109) are wider. The tight lymphocyte QC limits are adequate for lymphocytosis, where lymphocyte numbers are generally increased well in excess of the QC limits. Unfortunately, the QC limits may be too wide for lymphopenia, monocytopenia and monocytosis. For example, a monocyte count >1 × 109/l is considered clinically significant, therefore, the possibility exists that a negative shift in the results will provide a false negative without violating the QC limits. Monocytes may be elevated in chronic infections and myeloproliferative/myelodysplastic disorders. However, we have other markers such as neutrophil and leukocyte number for infectious processes. Patients with myeloproliferative disorders do not typically present with isolated monocytosis but also demonstrate other cytosis, distinctive cytologic and histologic features, phenotypic changes on flow cytometry and abnormal clinical findings. Therefore, the possibility of clinical significant error appears to be remote.The QC limits for Hct and MPV appear to approximate the physiologic limits. Unlike Hgb, clinicians generally do not follow Hct serially; however, values outside the reference range elicit attention. Generally, values <0.3 are considered to be clinically significant, which is well below the lower limit of the normal reference range . Therefore, deviations well in excess of the QC limits ( ± 0.014) are required before clinical intervention is required. For MPV, an uncommonly ordered and rarely reported parameter, the clinically significant changes appear to be at least twice as much as the physiologic limits  instrumentation.It is probable that other hematology analyzers are capable of such precise analytic performance. In order for the hematology laboratory to totally embrace expanded QC limits, manufacturers must make available their instruments’ usual imprecision’s as well as well as their instruments’ poor imprecision’s (e.g. the 15P performance). Selection of control rules based on average performance can be misleading; after all, half the analyzers will deliver less than average performance. Use of these expanded rules should markedly reduce the needless re-analysis based on 1


Lactic acid at sufficiently acidic pH is a potent microbicide, and lactic acid produced by vaginal lactobacilli may help protect against reproductive tract infections. However, previous observations likely underestimated healthy vaginal acidity and total lactate concentration since they failed to exclude women without a lactobacillus-dominated vaginal microbiota, and also did not account for the high carbon dioxide, low oxygen environment of the vagina. Fifty-six women with low (0-3) Nugent scores  and no symptoms of reproductive tract disease or infection, provided a total of 64 cervicovaginal fluid samples using a collection method that avoided the need for sample dilution and rigorously minimized aerobic exposure. The pH of samples was measured by microelectrode immediately after collection and under a physiological vaginal concentration of CO2. Commercial enzymatic assays of total lactate and total acetate concentrations were validated for use in CVF, and compared to the more usual HPLC method. The average pH of the CVF samples was 3.5 ± 0.3 (mean ± SD), range 2.8-4.2, and the average total lactate was 1.0% ± 0.2% w/v; this is a five-fold higher average hydrogen ion concentration (lower pH) and a fivefold higher total lactate concentration than in the prior literature. The microbicidal form of lactic acid (protonated lactic acid) was therefore eleven-fold more concentrated, and a markedly more potent microbicide, than indicated by prior research. This suggests that when lactobacilli dominate the vaginal microbiota, women have significantly more lactic acid-mediated protection against infections than currently believed. Our results invite further evaluations of the prophylactic and therapeutic actions of vaginal lactic acid, whether provided in situ by endogenous lactobacilli, by probiotic lactobacilli, or by products that reinforce vaginal lactic acid. Trichomonas vaginalis  no obvious metabolic disturbances” , “normalginal pH ,45. Valu2) and low oxygen (O2) levels that prevail in the vaginal environment. The cervicovaginal partial pressure of CO2 is equivalent to systemic levels (38 mm Hg or 5%)[2 is rapidly lost when the vaginal lumen or a CVF sample is exposed to air . Loss of CO2 from CVF samples would increase the pH and lead to an overestimate of the in vivo value. Furthermore, the cervicovaginal partial pressure of O2 is in the hypoxic range of 4-14 mm Hg (2%), with only transient increases due to insertion of a tampon [2 160 mm Hg or 21%), many strains of lactobacilli preferentially produce acetic acid [consume previously formed lactic acid to do so [Second, almost all previous studies ignored the high carbon dioxide (COHg or 5%); this COa tampon  or diapha tampon , sexual a tampon  and, pretic acid , and somto do so . Measure2 loss, and 4) use analysis methods that minimally perturb the biochemical composition of the samples. The data we report here were generated using strict inclusion criteria and a quantitative evaluation of participants’ vaginal microbiotas via Nugent scoring; we measured pH, total lactate and total acetate concentrations in ex vivo  CVF samples, with prevention of or correction for the effect of CO2 loss. In addition, to compare our results with those of earlier investigations, measurements were also made under the aerobic and hypocapnic conditions typical of previous studies. An accurate assessment of the protection that can be provided by vaginal lactic acid therefore requires observations of pH and total lactate concentration that 1) include only participants with an LMD vaginal microbiota, 2) collect CVF samples with minimal aerobic exposure and make measurements under hypoxic conditions, 3) prevent or compensate for the alkalizing effect of COUnless otherwise stated, all reagents were supplied by Sigma-Aldrich Inc. .Each participant gave written informed consent at every sample collection; the study and study protocols were approved by the Johns Hopkins University Homewood Institutional Review Board on the Use of Human Subjects.Participants were required to be between 18 and 45 years old, in good general health, at least three days past the end of the most recent menstruation and most recent unprotected penile-vaginal intercourse, at least three weeks past the most recent use of vaginal or systemic antibiotics or antifungals, and free from vaginal symptoms . 2 = 6.0±0.7 mm Hg as measured with an MI-730 oxygen electrode, Microelectrodes Inc., Bedford NH, and partial pressure of CO2 = 38±2 mm Hg as analyzed by supplier), or provided a hypoxic and hypocapnic condition . There were no transport or storage steps in the protocol; participants inserted and removed the Softcup in a room adjacent to the prepared glove-box, and the cup coated with CVF was transferred to its new environment within seconds. Cervicovaginal fluid (CVF) was self-sampled at the laboratory using the non-absorbent disposable Softcup™ menstrual device  , therebyThe pH of the CVF clinging to the Softcup (still in the open centrifuge tube) was immediately measured inside the glove-box, using an MI-415-6 cm combination pH electrode (Microelectrodes Inc.) designed to measure pH in small volumes or thin layers of fluid, and a digital pH meter . With the possibility of heterogeneity within individual samples, we made three pH measurements in quick succession at different sites on each Softcup; however, the three replicate measurements in each CVF sample never varied by more than ±0.02 pH units, and only the mean of each set of three measurements is reported here. The time elapsing between the initial insertion of the Softcup in the vagina and the completion of all three pH measurements was less than one minute in all cases. 2 from CVF, additional samples (still on the Softcup inside the open centrifuge tube) were placed in an aerobic and hypercapnic incubator  instead of the glove-box, and allowed to equilibrate overnight. Each sample was then brought into room air, the pH microelectrode immediately placed in the sample, and pH monitored over the next ten minutes to determine the time-course of alkalinization due to loss of CO2. To observe the time-course of the alkalization caused by loss of COg  to collect the CVF from the Softcup. The centrifuge tube was returned to the glove-box, opened, and the Softcup removed and discarded. The tube was weighed and the mass of the sample was calculated by reference to the pre-use recorded weight of the tube. After removing sufficient CVF for analysis of total lactate and total acetate concentrations, a sterile cotton swab was dipped in the remaining CVF, rolled out on a microscope slide, and the slide air-dried for Gram-staining and Nugent scoring according to a standard protocol [After pH measurement, the centrifuge tube was sealed while still in the glove-box to maintain hypoxic conditions during centrifuging for one minute at 500protocol . Both chromatographic and enzymatic methods were used to measure total lactate and total acetate concentrations. Enzymatic analyses used the D-lactic acid/L-lactic acid Enzymatic BioAnalysis/Food Analysis UV method kit and the Acetic acid Enzymatic BioAnalysis/Food Analysis UV method kit . Unlike the more generally used HPLC method, these enzymatic assays do not require extensive dilution, purification, or other manipulation of the samples. Additionally, the assay reactions and incubations can be carried out in a glove-box, and only removed immediately before the spectrophotometric measurements are made. The time elapsing between removal from our hypoxic glove-box and completion of the spectrophotometric measurements was less than one minute in all cases. To mimic procedures used in numerous previous studies, a second cotton swab was dipped in eight CVF samples (in addition to the one used to prepare the microscope slide) and subjected to conditions and processes typical of swabs collected at a clinical site and transported to a laboratory for HPLC analysis. Briefly, each swab-tip was exposed to air and extracted by vortexing in 1.0 mL distilled water. The resulting fluid was centrifuged and syringe-filtered, following . The HPLA set of known-concentration standard solutions of acetate and lactate were prepared . These concentration standards were analyzed in control experiments and in association with every CVF sample to ensure consistency and comparability of data collected by different methods or under different conditions. CVF  and CVF extracts  were serially diluted with clinical saline and the entire series assayed, guaranteeing at least two data points falling within the dynamic range of each method for every participant-sample. Correcting for the individual dilution-factors within the series typically yielded similar but not identical values for the original sample-concentration; the reported values for original sample-concentrations are therefore the mean of at least two data points falling within the dynamic range of the indicated method.t test (comparisons were unpaired unless otherwise indicated); p values ≤ 0.05 were considered to be statistically significant. Statistical analysis was performed using PHStat2 version 3.0 (Microsoft Excel add-on). Results are reported as mean ± standard deviation of at least eight independently repeated observations . For calculating means, samples with undetectable analytes were assigned values equal to half the detection threshold. The difference between two means was tested using a two-tailed Student’s n = 23), Black (n = 17), or Asian (n = 19). In all, 67 CVF samples were collected; three samples were excluded due to Nugent score >3, and one due to leucorrhea (>10 polymorphonuclear leukocytes per high powered field) [A total of 59 participants were recruited, primarily from among the students and staff of the university; they were between 19 and 44 years old (mean age 23±6 years), and self-identified as non-Hispanic White (d field) . The low2 and CO2); the mean pH was 3.74±0.29 (range 3.15-4.18) (2 and ~ 5% CO2 in N2), mimicking the vaginal environment, and the mean pH was significantly lower at 3.48±0.18  . Each sample was then exposed to ambient air and the pH monitored over time. The mean initial pH (measured after ~10 sec exposure) was 3.53±0.23 (p = 0.01) but not significantly different from the mean of the samples measured in the hypoxic and isocapnic condition. The pH of the samples exposed to air increased by approximately 0.3 pH units within 3 minutes . Control experiments using the standard lactate and acetate solutions showed the enzymatic method was not affected by aerobic versus hypoxic or isocapnic versus hypocapnic conditions (data not shown). To rule out possible interference in the enzymatic method, several CVF samples were serially diluted with clinical saline and a known, constant amount of lactate and acetate added to all the dilutions. The dilutions were then analyzed by the enzymatic method; all dilutions showed a constant and appropriate increase in signal, compared to dilutions without added lactate or acetate (data not shown).The mean total lactate concentration of 48 freshly collected, hypoxically maintained CVF samples analyzed enzymatically was 1.0±0.2% (111±22 mM), range 0.72-1.48%. In contrast, acetate was undetectable (detection threshold of assay 0.0003%) in 44 samples by enzymatic assay. The four samples with detectable acetate ranged between 0.0025% and 0.028%.R2 = 0.91), further supporting lactic acid as the primary determinant of vaginal acidity when lactobacilli dominate the vaginal microbiota. The tight correlation we observe between total lactate concentration and pH also indicates that the permeability ratio of the vaginal epithelium for lactate and hydrogen ions is similar among individuals. We note that in 2 (open circles vs filled circles) compared to the lesser increase in pH observed at higher total lactate concentration, presumably since buffer capacity is proportional to total lactate. In addition, we found no correlation between sample pH or total lactate concentration with sample mass, or with Nugent score (within the studied 0-3 range), and there was no significant difference between the means of total lactate measured with or without CO2 . We found a strong inverse linear relationship between the total lactate concentration and the pH of CVF (p = 0.001) when assayed both by HPLC and by the enzymatic method. Moreover, acetate was detectable in only one of eight samples by enzymatic analysis before preparation for HPLC but was measurable in seven of these eight samples after preparation for HPLC with a mean concentration of 0.17±0.09%. Preparation of samples for HPLC analysis caused a greater than twenty-fold increase in acetic acid concentration to levels consistent with those reported in the prior literature. Eight additional CVF samples  were processed for HPLC analysis. Processing samples for HPLC significantly decreased total lactate to 0.63±0.23%  vaginal microbiota, and reports measurements made in CVF samples collected without dilution or other modification, with rigorously minimized exposure to air, as well as ameliorating and correcting for the effect of CO2 levels in samples during pH measurements  being totally inactive [in vivo microbicidal action of vaginal lactic acid when lactobacilli dominate the vaginal microbiota may be significantly more protective than previously thought. Although an ejaculate transiently alkalinizes the vagina after unprotected intercourse, reducing the protective actions of vaginal acidity against male-to-female transmission, the level of vaginal lactic acid we document here is likely to reduce female-to-male transmission of vaginally shed acid-sensitive pathogens. Together, the higher concentrations of hydrogen ions and total lactate that we determined here indicate an 11-fold higher concentration of protonated lactic acid, LAH: based on the pKa of lactic acid and the Henderson Hasselbalch relation, the mean LAH concentration in our samples was 0.69% (76 mM), compared to an LAH concentration of 0.06% (7 mM) when calculated from the literature values of vaginal pH and total lactate concentration. This is a critical difference in terms of microbicidal activity, since we have shown that it is only LAH that inactivates HIV, with the lactate anion , and probiotic strains of lactobacilli are being developed that might enable these women to maintain an LMD vaginal microbiota. Currently, the ability to produce H