Non-genotoxic Restoration of the Hematolymphoid System in Fanconi Anemia.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients with many different blood and immune diseases; however, current treatment regimens contain non-specific chemotherapy and/or irradiation conditioning, which carry both short-term and long-term toxicities. The use of such agents may be particularly harmful for patients with Fanconi anemia (FA), who have genetic mutations resulting in deficiencies in DNA repair, leading to increased sensitivity to genotoxic agents. mAb-based conditioning has been proposed as an alternative conditioning strategy for HSCT that minimizes these toxicities by eliminating collateral tissue damage. Given the high need for improved treatments for FA patients, we aimed to evaluate the efficacy of different αCD117 mAb agents and immunosuppression on hematopoietic stem cell (HSC) depletion and explored their ability to safely establish therapeutic donor hematopoiesis post-HSCT in FA disease models. We evaluated the effects of different concentrations of αCD117 mAbs in vitro and in vivo on HSC growth and depletion. To further assess the efficacy of mAb-based conditioning, Fancd2-/- animals were treated with αCD117 mAb and combination agents with αCD47 mAb and antibody-drug-conjugates (ADCs) for syngeneic HSCT. Immunosuppression αCD4 mAb was added to all in vivo experiments due to a slightly mismatched background between the donor grafts and recipients. Immunosuppressant cocktails were also given to Fancd2-/- animals to evaluate the efficacy of mAb-based conditioning in the haploidentical setting. Statistical analyses were done using the unpaired t-test. We found that antagonistic αCD117 mAbs alone do not deplete host HSCs or enhance HSCT effectively in FA mouse models; however, the potency of αCD117 mAbs can be safely augmented through combination with αCD47 mAbs and with ADCs, both of which lead to profound HSC depletion and establishment of long-term donor engraftment post-syngeneic HSCT. This is the first time these approaches have been tested in parallel in any disease setting, with the greatest donor engraftment observed after CD117-ADC conditioning. Interestingly, our data also suggest that HSC-targeted conditioning is not necessary in HSCT for FA, as high donor HSC engraftment was observed with mAb-based immune suppression alone with immunologically matched and mismatched haploidentical grafts. These results demonstrate the safety and efficacy of several different non-genotoxic mAb-based conditioning strategies in the FA setting. In addition, they show that if sufficient immunosuppression is given to obtain initial donor HSC engraftment, turnover of a majority of the hematolymphoid system can result, likely owing to the survival advantage of wild-type HSCs over FA HSCs. Such non-toxic all-mAb-based conditioning strategies could be transformative for FA patients and those with other hematolymphoid diseases.

Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals.

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

Short communication: Effect of feeding pooled and nonpooled high-quality colostrum on passive transfer of immunity, morbidity, and mortality in dairy calves.

Pooling colostrum is commonly practiced on Irish dairy farms. Pooling can result in dilution when colostrums with high and low IgG concentrations are mixed, thereby predisposing calves to failure of passive immunity. The objectives of this study were to compare IgG concentrations in colostrum from individual cows with colostrum pooled from several cows, and assess serum IgG concentrations, morbidity, and mortality among calves fed colostrum from their own dam, from a different cow, or pooled from several cows. We hypothesized that pooling colostrum reduces IgG concentration due to dilution compared with colostrum from individual cows, and that calves fed pooled colostrum achieve lower serum IgG concentrations than calves fed colostrum from individual cows. Calves were randomly assigned to 1 of 3 groups: (1) fed colostrum from their own dam (n = 20); (2) fed colostrum from a different dam (n = 20); or (3) fed pooled colostrum (n = 18). A sample of colostrum fed to each calf was collected. Serum samples were collected from calves at birth (0 h) and at 24 h after colostrum feeding. Colostrum and serum IgG concentrations were measured by radial immunodiffusion. Calves were weighed at birth and at weaning, and the health status of each calf was assessed twice daily. Health assessment was based on general demeanor, rectal temperature, fecal consistency, respiratory rate, and the presence of cough, nasal, or ocular discharge. Colostrum and serum IgG concentrations, and weaning weights were compared using ANOVA. Associations between group and morbidity or mortality rates were compared using χ2 or Fisher's exact tests. Median and 95% confidence intervals (95% CI) of IgG concentrations of colostrum were 99.4 (81.8-111.5), 95.2 (84.1-107.2), and 100.7 (90.5-104.4) g/L for own dam, different dam, and pooled groups, respectively. We did not find any differences in colostrum IgG concentrations among the colostrum sources. Median (95% CI) serum IgG concentrations at 24 h were 52.0 (45.6-65.9), 55.7 (51.2-65.9), and 53.1 (46.2-63.7) g/L for calves that received colostrum from own dam, different dam, and pooled, respectively. All calves achieved adequate passive immunity. Serum IgG concentrations at 24 h, weaning weights, and proportions of morbidity and mortality were not different among the 3 groups. Our results suggest that on dairy farms where median colostrum IgG concentrations are high and colostrum management is optimal, pooling has a minimal effect on passive immunity and subsequent calf health.

Short communication: Use of a digital refractometer in assessing immunoglobulin G concentrations in colostrum and the first 5 transition milkings in an Irish dairy herd.

Transition milk is a source of immunoglobulin G (IgG) and could potentially be used to provide calves with passive immunity, when the IgG concentration is ≥50 g/L. Assessment of IgG concentrations in transition milk would be required before feeding and could be conducted using cow-side tests such as refractometers. Currently, limited information is available on the ability of refractometers to assess transition milk quality. We hypothesized that digital refractometry could be used to provide an accurate cow-side assessment of IgG concentrations in colostrum and transition milk, and IgG concentration in colostrum and one or more transition milking in an Irish herd is >50 g/L. The objectives of this study were to determine the IgG concentrations in colostrum and first, second, third, fourth, and fifth transition milk, and determine the utility of a digital refractometer in assessing quality of colostrum and transition milk produced by cows in a pasture-based dairy production system. A convenient sample of 75 dairy cows were enrolled. Colostrum and transition milk IgG concentrations were determined by radial immunodiffusion and refractometry. Sensitivity and specificity of the refractometer were determined and cut-off points that maximized sensitivity and specificity were determined using receiver operating characteristic curves. Median (range) IgG concentrations in colostrum and first, second, third, fourth, and fifth milking were 99.6, 43.5, 12.5, 5.3, 1.9, and 1.8 g/L, respectively. The sensitivity (0.8-1) of digital refractometry in identifying samples with low IgG concentrations in colostrum, first, second, and third transition milk was acceptable. In contrast, digital refractometry was not useful for assessing IgG concentrations in the fourth and fifth milking due to low IgG concentrations.

Randomized controlled clinical trial on the effect of oral immunoglobulin supplementation on neonatal dairy calves with diarrhea.

BACKGROUND: Nonantibiotic alternatives providing local gut immunity have been recommended for managing calf diarrhea. ANIMALS: One hundred and two calves with diarrhea. HYPOTHESIS: Oral supplementation with immunoglobulins in calves with diarrhea will reduce time to resolution of diarrhea, number of treatment events, and mortality rate. METHODS: Randomized controlled trial. Calves were assigned into 1 of 3 groups. The treatment group was supplemented with 20 g of immunoglobulins in milk twice daily for 14 days. The placebo group was supplemented with 20 g of a product with similar nutritional value as the treatment group, but without immunoglobulins, in milk, twice daily for 14 days. The control group received no supplements. Medical treatments, time to resolution of diarrhea, and case fatality rates were compared. RESULTS: There was no difference in the proportion of treatment events (treatment, 79% versus placebo, 77% versus control, 71%) among groups (P = .69). The median time to resolution of diarrhea was not different between the treatment (10.5 days; 95% confidence interval [CI], 7, 13) and control (8 days; 95% CI, 5, 10) groups (P = .08) or between the placebo (6.5 days; 95% CI, 3, 9) and control groups (P = .89). Median time to resolution was shorter (P = .008) in the placebo compared to the treatment group (6.5 versus 10.5 days). Case fatality rates among groups (treatment, 12% versus placebo, 3% versus control, 3%) were not different (P = .36). CONCLUSIONS AND CLINICAL IMPORTANCE: Expected benefits of conferring local gut immunity by immunoglobulin supplementation in calves with diarrhea were not evident.

Thermographic evaluation of primary closure and second intention healing in dairy calves.

OBJECTIVE: To evaluate the suitability of infrared thermography in assessing healing of surgically created wounds that are managed by primary closure or second intention in neonatal dairy calves during a 3-week period. STUDY DESIGN: Randomized clinical trial. ANIMALS: Six Jersey bull calves. METHODS: Two skin patches approximately 10 cm2 were shaved on each hind limb of all calves. The dorsal patch was designated the wound creation site, and the ventral patch was the control. The wound creation sites were randomly assigned for either primary closure or healing by second intention. Wounds were created by using an 8-mm biopsy punch. Thermographic imaging was performed prior to wound creation and at 0 minutes; 15 minutes; 2, 4, 8, 12, and 24 hours; and 2, 3, 4, 7, 10, 14, and 21 days postwounding. RESULTS: There were no differences in skin temperature changes observed between wounds that were managed by primary closure or second intention (P = .9934) at any time. Time after wound creation had an effect on the skin temperature (P < .0001), with skin temperature consistently warmer (P < .05) 2, 4, and 8 hours after creation of wounds compared with subsequent times. CONCLUSION: Infrared thermography was unable to detect differences in wound healing by primary closure or second intention in this model. CLINICAL SIGNIFICANCE: Thermographic monitoring to detect differences in wound healing was not evident in this model. This model might be useful in monitoring temporal changes during early wound repair.

VALIDATION OF IMMUNOCRIT AS A DIAGNOSTIC TEST TO DETECT FAILURE OF PASSIVE TRANSFER OF IMMUNITY IN HAND-REARED NONDOMESTIC RUMINANT NEONATES.

Domestic and nondomestic ruminant neonates with failure of passive transfer of immunity (FPI) have an increased risk of morbidity and mortality. Hand-reared individuals often have a higher incidence of FPI compared with those that are dam-reared. Hand-reared, nondomestic ruminant neonates raised at zoological establishments are often fed a commercially available bovine colostrum replacer. Consequently, it is desirable to monitor for FPI using a single test that performs well across a wide range of species. The immunocrit test method has been reported to have acceptable diagnostic sensitivity for detecting FPI in dairy calves. Advantages of the immunocrit are that it is inexpensive, quick, easy to perform, non-species-specific, and only precipitates immunoglobulins. This study evaluated the ability of immunocrit to predict serum immunoglobulin levels and FPI via comparison with radial immunodiffusion (reference method). The study used 100 serum samples collected from hand-reared ruminant neonates from 29 nondomestic species, 12-120 hr old, that were fed a commercial bovine colostrum replacer at a zoological institution. Sensitivity, specificity, and likelihood ratios for FPI were determined using receiver operating characteristic (ROC) curves. The immunocrit cutoff point of 14% maximized sensitivity at 88.6% (95% CI, 78.7-94.9%) and specificity at 83.3% (95% CI, 65.3-94.4%). The area under the ROC curve was 0.92 (95% CI, 0.87-0.98; P < 0.001). This research demonstrates that the immunocrit test has acceptable performance in detecting FPI in a wide variety of nondomestic hand-reared ruminant neonates. Use of the immunocrit test will help improve the identification of FPI neonates and aid in the management of colostrum feeding practices of nondomestic ruminant species in captivity.

Effect of intravenous plasma transfusion on granulocyte and monocyte oxidative and phagocytic activity in dairy calves with failure of passive immunity.

Plasma administration has been recommended in calves older than 48h with failure of passive immunity (FPI) to provide immunity consistent with adequate colostral ingestion. However, the protective serum immunoglobulin G (IgG) concentrations (≥1000mg/dL) of plasma derived IgG only lasts up to 12h. In addition to IgG, maternally derived colostral cells also confer immunity. The objective of the study was to determine the effect of intravenous plasma transfusion on granulocyte and monocyte oxidative and phagocytic activity in calves with FPI. Twenty-seven, one day-old, Jersey calves were assigned into 3 groups. The colostral (CL, N=9) group received 3L of colostrum once by oroesophageal tubing. Two other groups of calves received 1L of colostrum once by oroesophageal tubing and were assigned based on their health status (sick or non-sick) at 4days of age, as the sick-group (SG, N=7) or the non-sick (NG, N=11) groups. At 4days of age, the SG and NG groups were administered plasma intravenously at 30mL/kg. Granulocyte and monocyte oxidative and phagocytic activity was determined by flow cytometry. There was no significant difference in the granulocyte and monocyte oxidative or phagocytic activity among the 3 groups (P>0.05). Plasma administration had no significant effect on the oxidative or phagocytic activity of granulocytes or monocytes. In clinical practice, plasma administration for enhancing oxidative or phagocytic activity of granulocytes or monocytes, alone, might not be justified in calves with FPI.