Syngeneic transplantation with peripheral blood mononuclear cells collected after the administration of recombinant human granulocyte colony-stimulating factor

Five syngeneic transplants were performed in four patients following myeloablative therapy using unmodified peripheral blood mononuclear cells (PBMCs) collected after the administration of recombinant human granulocyte colony stimulating factor (rhG-CSF) to normal donors. The only toxicity experienced by the four normal donors was bone pain. Four patients received two collections of PBMCs, and a second transplant was performed in one patient with one collection. The patients received a median of 20.53 x 10(8) total nucleated cells/kg (range 20 to 25.5), 11.3 x 10(8) total mononuclear cells/kg (range 6.52 to 17.2), 113.1 x 10(4)/kg CFU-GM (range 46.7 to 211.8) and 9.6 x 10(6) CD34+ cells/kg (range 1.6 to 12.6) Post-transplant growth factors were not administered. The median time to an absolute neutrophil count greater than 0.5 x 10(9)/L was 14 days (range 10 to 18). The median time to platelet transfusion independence was 11 days (range 10 to 13). Two patients had the number of CD3+ T lymphocytes determined in the pheresis product. An average of 3.04 x 10(10) CD3+ cells were collected per pheresis. This represents an approximate 1 log increase over the number of T lymphocytes in a typical bone marrow transplant. Rh-GCSF can be used to mobilize peripheral blood progenitor cells from normal donors with minimal toxicity. Studies of allogeneic transplants using PBMCs collected after rhG-CSF administration to determine permanent grafting ability and the incidence and severity of graft-versus-host disease are warranted.     

Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation

Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death.     

Oral ganciclovir in children: pharmacokinetics, safety, tolerance, and antiviral effects. The Pediatric AIDS Clinical Trials Group

The pharmacokinetics, safety, tolerance, and antiviral effects of ganciclovir (Gcv) administered orally were evaluated in 36 children infected with cytomegalovirus (CMV) who were severely immunocompromised by infection with human immunodeficiency virus type 1. In this dose-escalation study, 30 mg/kg of Gcv administered every 8 h produced serum levels similar to the dose (1 g/8 h) effective for maintenance treatment of CMV retinitis in adults. In older children, serum Gcv concentrations were similar after the administration of capsules and suspension. All doses (10-50 mg/kg/8 h) studied were safe and, except for the volume of suspension or number of pills, were well tolerated. Oral Gcv was associated with a decrease in the detection of CMV by culture or polymerase chain reaction. CMV disease occurred in 3 children during the study: one developed Gcv resistance, another had harbored resistant virus at study entry, and a third had wild-type CMV     

Ovine forestomach matrix biomaterial is a broad spectrum inhibitor of matrix metalloproteinases and neutrophil elastase

Proteases play a critical role in the ordered remodelling of extracellular matrix (ECM) components during wound healing and tissue regeneration. However, the usually ordered proteolysis is compromised in chronic wounds due to over‐expression and high concentrations of matrix metalloproteinase's (MMPs) and neutrophil elastase (NE). Ovine forestomach matrix (OFM) is a decellularised extracellular matrix‐based biomaterial developed for tissue regeneration applications, including the treatment of chronic wounds, and is a heterogeneous mixture of ECM proteins and proteoglycans that retains the native structural and functional characteristics of tissue ECM. Given the diverse molecular species present in OFM, we hypothesised that OFM may contain components or fragments that inhibit MMP and NE activity. An extract of OFM was shown to be a potent inhibitor of a range of tissue MMPs (IC₅₀s = 23 ± 5 to 115 ± 14 µg/ml) and NE (IC₅₀ = 157 ± 37 µg/ml), and was more potent than extracts prepared from a known protease modulating wound dressing. The broad spectrum activity of OFM against different classes of MMPs (i.e. collagenases, gelatinases and stromelysins) may provide a clinical advantage by more effectively addressing the protease imbalance seen in chronic wounds.     

Capillary transit time heterogeneity and flow-metabolism coupling after traumatic brain injury

Most patients who die after traumatic brain injury (TBI) show evidence of ischemic brain damage. Nevertheless, it has proven difficult to demonstrate cerebral ischemia in TBI patients. After TBI, both global and localized changes in cerebral blood flow (CBF) are observed, depending on the extent of diffuse brain swelling and the size and location of contusions and hematoma. These changes vary considerably over time, with most TBI patients showing reduced CBF during the first 12 hours after injury, then hyperperfusion, and in some patients vasospasms before CBF eventually normalizes. This apparent neurovascular uncoupling has been ascribed to mitochondrial dysfunction, hindered oxygen diffusion into tissue, or microthrombosis. Capillary compression by astrocytic endfeet swelling is observed in biopsies acquired from TBI patients. In animal models, elevated intracranial pressure compresses capillaries, causing redistribution of capillary flows into patterns argued to cause functional shunting of oxygenated blood through the capillary bed. We used a biophysical model of oxygen transport in tissue to examine how capillary flow disturbances may contribute to the profound changes in CBF after TBI. The analysis suggests that elevated capillary transit time heterogeneity can cause critical reductions in oxygen availability in the absence of ‘classic'' ischemia. We discuss diagnostic and therapeutic consequences of these predictions.     

EMC10 homozygous variant identified in a family with global developmental delay,mild intellectual disability,and speech delay

In recent years, several genes have been implicated in the variable disease presentation of global developmental delay (GDD) and intellectual disability (ID). The endoplasmic reticulum membrane protein complex (EMC) family is known to be involved in GDD and ID. Homozygous variants of EMC1 are associated with GDD, scoliosis, and cerebellar atrophy, indicating the relevance of this pathway for neurogenetic disorders. EMC10 is a bone marrow-derived angiogenic growth factor that plays an important role in infarct vascularization and promoting tissue repair. However, this gene has not been previously associated with human disease. Herein, we describe a Saudi family with two individuals segregating a recessive neurodevelopmental disorder. Both of the affected individuals showed mild ID, speech delay, and GDD. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify candidate genes. Further, to elucidate the functional effects of the variant, quantitative real-time PCR (RT-qPCR)-based expression analysis was performed. WES revealed a homozygous splice acceptor site variant (c.679-1G>A) in EMC10 (chromosome 19q13.33) that segregated perfectly within the family. RT-qPCR showed a substantial decrease in the relative EMC10 gene expression in the patients, indicating the pathogenicity of the identified variant. For the first time in the literature, the EMC10 gene variant was associated with mild ID, speech delay, and GDD. Thus, this gene plays a key role in developmental milestones, with the potential to cause neurodevelopmental disorders in humans.     

Newborn Screening for SCID in New York State: Experience from the First Two Years

Purpose

To describe the process and assess outcomes for the first 2 years of newborn screening for severe combined immunodeficiency (SCID NBS) in New York State (NYS).

Methods

The NYS algorithm utilizes a first-tier molecular screen for TRECs (T-cell receptor excision circles), the absence of which is indicative of increased risk of immunodeficiency.

Results

During the first 2 years, 485,912 infants were screened for SCID. Repeat specimens were requested from 561 premature and 746 non-premature infants with low or borderline TRECs. A total of 531 infants were referred for diagnostic evaluation leading to identification of 10 infants with SCID and 87 with a clinically significant non-SCID abnormality based on flow cytometry or CBC results (positive predictive value 20.3 %). Nine infants were diagnosed with typical SCID and one with leaky SCID. SCID diagnoses included two patients with adenosine deaminase deficiency, three patients with typical and one with leaky IL2RG-related SCID, one patient with IL7Rα-related SCID, and three cases of typical SCID, etiology unknown. TRECs were undetectable in eight of the nine babies with typical SCID. Infants with other non-SCID conditions included 27 patients with a syndrome that included T-cell impairment, 18 of which had DiGeorge syndrome. Seventeen infants had T-cell impairment secondary to another clinically significant condition, and 13 were classified as ‘other’. Among 30 infants classified as idiopathic T-cell lymphopenia, 11 have since resolved, and the remainder continues to be followed. One infant with undetectable TRECs had normal follow-up studies. Molecular studies revealed the presence of two changes in the infant’s DNA.

Conclusions

Overall, ten infants with SCID were identified during the first 2 years of screening in NYS, yielding an incidence of approximately 1 in 48,500 live births, which is consistent with the incidence observed by other states screening for SCID. The incidence of any clinically significant laboratory abnormality was approximately 1 in 5,000; both estimates are higher than estimates prior to the onset of newborn screening for SCID. Improvements to the NYS algorithm included the addition of a borderline category that reduced the proportion of infants referred for flow cytometric analysis, without decreasing sensitivity. We identified a large number of infants with abnormal TRECs and subsequent idiopathic T-cell lymphopenia. Long-term follow-up studies are needed to determine the prognosis and optimal treatment for this group of patients, some of whom may present with previously unrecognized, transient lymphopenia of infancy.