Structural Characterization of the D179N and D179Y Variants of KPC-2 ß-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these ß-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the ß-lactam/ß-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 ß-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the ß-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility.

Clinical predictors of donor antibody titre and correlation with recipient antibody response in a COVID-19 convalescent plasma clinical trial.

BACKGROUND: Convalescent plasma therapy for COVID-19 relies on transfer of anti-viral antibody from donors to recipients via plasma transfusion. The relationship between clinical characteristics and antibody response to COVID-19 is not well defined. We investigated predictors of convalescent antibody production and quantified recipient antibody response in a convalescent plasma therapy clinical trial. METHODS: Multivariable analysis of clinical and serological parameters in 103 confirmed COVID-19 convalescent plasma donors 28 days or more following symptom resolution was performed. Mixed-effects regression models with piecewise linear trends were used to characterize serial antibody responses in 10 convalescent plasma recipients with severe COVID-19. RESULTS: Donor antibody titres ranged from 0 to 1 : 3892 (anti-receptor binding domain (RBD)) and 0 to 1 : 3289 (anti-spike). Higher anti-RBD and anti-spike titres were associated with increased age, hospitalization for COVID-19, fever and absence of myalgia (all P < 0.05). Fatigue was significantly associated with anti-RBD (P = 0.03). In pairwise comparison amongst ABO blood types, AB donors had higher anti-RBD and anti-spike than O donors (P < 0.05). No toxicity was associated with plasma transfusion. Non-ECMO recipient anti-RBD antibody titre increased on average 31% per day during the first three days post-transfusion (P = 0.01) and anti-spike antibody titre by 40.3% (P = 0.02). CONCLUSION: Advanced age, fever, absence of myalgia, fatigue, blood type and hospitalization were associated with higher convalescent antibody titre to COVID-19. Despite variability in donor titre, 80% of convalescent plasma recipients showed significant increase in antibody levels post-transfusion. A more complete understanding of the dose-response effect of plasma transfusion amongst COVID-19-infected patients is needed.

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Insulin-like growth factor I (IGF-I) stimulates hematopoiesis. We examined whether bone marrow stromal cells synthesize IGF-I. Secretion of IGF-I immunoreactivity by cells from TC-1 murine bone marrow stromal cells was time-dependent and inhibited by cycloheximide. Gel filtration chromatography under denaturing conditions of TC-1 conditioned medium demonstrated two major peaks of apparent IGF-I immunoreactivity with molecular weights of approximately 7.5-8.0 kD, the size of native IGF-I, and greater than 25 kD. Expression of IGF-I mRNA was identified by both RNase protection assay and reverse transcription/polymerase chain reaction. To determine whether the greater than 25-kD species identified by RIA possessed IGF-binding activity, a potential cause of artifactual IGF-I immunoreactivity, charcoal adsorption assay of these gel filtration fractions was performed. The peak of IGF-binding activity coeluted with apparent IGF-I immunoreactivity suggesting that TC-1 cells secrete IGF-binding protein(s). Unfractionated conditioned medium exhibited linear dose-dependent increase in specific binding of [125I]-IGF-I with a pattern of displacement (IGF-I and IGF-II much greater than insulin) characteristic of IGF-binding proteins. Western ligand analysis of conditioned medium showed three IGF-I binding species of approximately 31, 38, and 40 kD. These data indicate that TC-1 bone marrow stromal cells synthesize and secrete IGF-I and IGF-binding proteins and constitute a useful model system to study their regulation and role in hematopoiesis.

Rehabilitation of hand in subacute tetraplegic patients based on brain computer interface and functional electrical stimulation: a randomised pilot study.

OBJECTIVE: To compare neurological and functional outcomes between two groups of hospitalised patients with subacute tetraplegia. APPROACH: Seven patients received 20 sessions of brain computer interface (BCI) controlled functional electrical stimulation (FES) while five patients received the same number of sessions of passive FES for both hands. The neurological assessment measures were event related desynchronization (ERD) during movement attempt, Somatosensory evoked potential (SSEP) of the ulnar and median nerve; assessment of hand function involved the range of motion (ROM) of wrist and manual muscle test. MAIN RESULTS: Patients in both groups initially had intense ERD during movement attempt that was not restricted to the sensory-motor cortex. Following the treatment, ERD cortical activity restored towards the activity in able-bodied people in BCI-FES group only, remaining wide-spread in FES group. Likewise, SSEP returned in 3 patients in BCI-FES group, having no changes in FES group. The ROM of the wrist improved in both groups. Muscle strength significantly improved for both hands in BCI-FES group. For FES group, a significant improvement was noticed for right hand flexor muscles only. SIGNIFICANCE: Combined BCI-FES therapy results in better neurological recovery and better improvement of muscle strength than FES alone. For spinal cord injured patients, BCI-FES should be considered as a therapeutic tool rather than solely a long-term assistive device for the restoration of a lost function.

Secretion and binding of insulin-like growth factor-I by a human medullary-thyroid carcinoma cell-line.

The TT human medullary thyroid carcinoma cell line secretes a 7.5 kD species with insulin-like growth factor (IGF-I) immunoreactivity and HPLC mobility and expresses IGF-I mRNA. Conditioned medium also contained IGF-binding activity with a pattern of displacement of [I-125]-IGF-I characteristic of IGF binding proteins. Western ligand analysis and immunobloting with anti-IGFBP4 antiserum identified a 34 kD IGF-I binding species. Northern analysis identified a 2.1 kb IGFBP4 mRNA species. Cell surface binding of [I-125]-IGF-I identified Type I IGF receptors. TT cells constitute a useful model to study the IGF-I autocrine system in the transformed state.

Current trends in community-acquired methicillin-resistant Staphylococcus aureus at a tertiary care pediatric facility.

BACKGROUND: The prevalence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections increased at the University of Chicago Children's Hospital (UCCH) from 10 per 100,000 admissions from 1988 to 1990 to 259 per 100,000 admissions from 1993 to 1995. Because this increase may have represented a one time occurrence or a limited disease outbreak, we updated our previous observations at UCCH in 1998 and 1999 to see whether this trend had continued. DESIGN: Prospective observational study. RESULTS: Twenty-three hospitalized children had an MRSA isolate during the 1-year study period. Ten were community-acquired, equally distributed between children with predisposing risk factors and those without. The overall prevalence of community-acquired MRSA was 208 per 100,000 admissions. Seven of the 10 community-acquired MRSA isolates were susceptible to clindamycin. Skin and soft tissue infections predominated among the children with a community-acquired MRSA isolate. Pulsed field gel electrophoresis of the 10 community-acquired MRSA isolates revealed 8 distinct patterns; these data suggest that multiple clones were circulating at UCCH. CONCLUSION: MRSA are no longer confined to children with established risk factors. The prevalence of community-acquired MRSA among children without identified risk factors is high in our institution.

Cesarean section does not improve outcome in gastroschisis.

Elective cesarean section (CS) following prenatal diagnosis of gastroschisis has been advocated to decrease morbidity and mortality. To examine this hypothesis, we reviewed the records of 28 consecutive patients with gastroschisis treated between 1975 and 1987. Fourteen infants were delivered vaginally (V) and fourteen by CS, of which seven were elective, five were for fetal distress, and two were for breech presentation. Prenatal diagnosis was made in nine infants in the CS group and none in the vaginal group. The two groups were comparable in gestational age (V = 37.6 weeks, CS = 35.8 weeks, P = .05), birth weight (V = 2,508 g, CS = 2,444 g, P = NS), and five-minute Apgar score (V = 7.8, CS = 6.8, P = NS). Outcome was similar as measured by hospital mortality (V = 0/14, CS = 1/14, P = NS), complications (V = 4/14, CS = 5/14, P = NS), days to enteral feeding (V = 14, CS = 19, P = NS), and days in the hospital (V = 27, CS = 34, P = NS). The only complication related to mode of delivery was preventable; an infant delivered vaginally had avulsion of a short segment of mesentery requiring bowel resection. Infants born by CS were slightly more likely to have primary closure (5/14) than babies delivered vaginally (3/14, P = NS), but this may reflect independent trends in the last 5 years. Elective CS following prenatal diagnosis in seven patients did not improve outcome; primary closure was achieved in only one infant, and three had a complication. Since these data show no significant difference in morbidity and mortality between vaginal and CS delivery, we suggest that CS should not be recommended simply because a prenatal diagnosis of gastroschisis is made.

Fetal hematopoietic stem cell transplantation into beta-thalassemic mice.

The transplantation of fetal-derived hematopoietic stem cells (HSCs) may potentially be used to treat hemoglobinopathies, immunodeficiencies, and storage diseases. The levels of donor cell engraftment needed to reconstitute the recipient's hematopoietic system are disease-dependent and remain unknown for most deficiencies. We have explored the application of fetal hematopoietic stem cell transplants for the amelioration of hemolytic disease in a murine model of beta-thalassemia. Nonirradiated neonatal homozygous beta-thalassemic mice were transplanted intraperitoneally (IP) with 10(6) fetal liver cells from syngeneic nonthalassemic murine fetal donors (14 to 16 days gestation). Donor hemoglobin was demonstrated in the peripheral blood of 9 of 14 transplant recipients at levels ranging from 8.8% to 27.1% at 30 days. The levels of engraftment in 6 of these 9 transplant chimeras remained stable or increased up to 150 days after transplantation, with levels ranging from 13.6% to 54.6% at 280 days. Three chimeras have demonstrated gradually decreasing engraftment after 200 days. The degree of engraftment correlated with clinically relevant improvement: decreased reticulocyte counts (8.4% to 15.7% in chimeras [n = 9] v 17.1% to 19.1% in controls [n = 8], P = .01), increased mean RBC deformability, and the significant reduction in extramedullary hematopoiesis and iron deposits seen on histological examination of chimeric liver and spleen. These data demonstrate that fetal HSC transplants results in significant long-term chimerism with favorable alterations in red cell characteristics, and decreased hemolytic anemia in beta-thalassemia.

Alimentary tract malignancies in children.

Alimentary tract malignancies in children are unusual. From 1952 to 1996, 54 patients (55 cases) underwent surgery at The Children's Hospital, Columbus for intestinal malignancy. Their records were reviewed retrospectively. The mean age at diagnosis was 9.3 years (range, 1 to 17 years). There were 35 boys and 19 girls (M:F ratio, 1:8). Mean follow-up was 108 months. Laparotomy was performed in all but one child. The primary tumor sites included the colon (31 cases), small bowel (12 cases), appendix (9 cases), and stomach (3 cases). Seventy-five percent of the tumors were non-Hodgkin's lymphomas (41 cases), followed by appendiceal carcinoid (9 cases), colon adenocarcinoma (3 cases), and gastric sarcoma (2 cases). Lymphoma occurred in 28 of 31 nonappendiceal large bowel tumors and was the only tumor type seen in the small intestine. There were 21 Burkitt's, 11 lymphoblastic, 6 small cell, and 5 large cell lymphomas. There was a statistically significant increase in the frequency of small bowel lymphoma after 1982 (10 of 20) in comparison with that before 1982 (2 of 21), P < .05. This coincided with an increase in Burkitt's lymphoma from 5 of 21 (pre-1982) to 16 of 20 (post-1982), and a decrease in lymphoblastic lymphomas from 7 of 21 (pre-1982) to 2 of 20 (post-1982), P < .05. Localized disease and complete resection favored survival in lymphoma, whereas age, sex, and urgency of operation had no influence on survival. This study highlights the predominance of lymphoma as the most common small and large bowel tumor and highlights the emergence of nonendemic Burkitt's as a major entity in pediatric intestinal malignancy.

Nonteratomatous germ cell tumors in children.

BACKGROUND/PURPOSE: Totipotential germ cells may give rise to a broad range of tumors. The teratomatous variety of germ cell tumors has been the subject of several large studies. The goal of the current study was to describe the clinical features of nonteratomatous germ cell tumors (NTGCT) by reviewing a large series of patients. METHODS: Between 1945 and 1997, there were 78 cases of nonteratomatous germ cell tumors (NTGCT's) in children at The Children's Hospital, Columbus. Their records were reviewed retrospectively. There were 35 boys and 43 girls (M:F ratio 0.8). Mean follow-up was 87 months. RESULTS: Histological subtypes included germinoma (33 cases, 42%), endodermal sinus tumor (24 cases, 31%), embryonal carcinoma (12 cases, 15%), gonadoblastoma (4 cases, 5%), mixed histology (4 cases, 5%), and choriocarcinoma (1 case, 2%). Forty-two tumors were in gonadal sites, but a significant percentage were extragonadal (36 cases, 46%). Forty-six patients (59%) had localized disease, 18 (23%) had regional disease, and 14 (18%) had metastases. Treatment consisted of surgery and selective chemotherapy and radiation. Complete tumor resection was more likely for gonadal (29 of 42, 69%) than extragonadal primaries (15 of 36, 41%; P < or = .05). Forty-nine (63%) of all patients survived, whereas 29 (37%) died of their disease. Survival in patients with gonadal primaries (32 of 42, 76%) exceeded that in patients with extragonadal primaries (17 of 36, 47%; P < or = .01). Survival in patients with localized disease (34 of 46, 74%) exceeded that in patients with regional extension or metastases (15 of 32, 47%; P < .05). CONCLUSIONS: This study highlights the fact that tumor location, gonadal versus extragonadal, was important in determining prognosis, whereas tumor histology was not. This may be the result of a higher rate of complete tumor resection for gonadal primaries and underscores the important role of surgery in the optimal treatment of these unusual tumors.

Violent injuries among women in an urban area.

The national statistics are familiar by now: each year, more than 2 million women are raped and/or physically assaulted; more than one-third of them are injured during their most recent assault. Annually, more than 500,000 women seek medical services as a result of violence-related injuries, often from hospital emergency departments. But national statistics cannot fully capture the extent of violence experienced by women in inner-city areas, nor do they point to modifiable risk factors at a community level. This Issue Brief highlights a new study that investigates the circumstances and correlates of violent injuries among women in one urban, low-income community.

A framework linkage map of bermudagrass (Cynodon dactylon x transvaalensis) based on single-dose restriction fragments.

This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps.

Extensive gastric heterotopia of the small intestine resulting in massive gastrointestinal bleeding, bowel perforation, and death: report of a case and review of the literature.

Gastric heterotopia of the small intestine is a rare occurrence outside of Meckel's diverticulum and intestinal duplication. The vast majority of cases of gastric heterotopia occur as polypoid or tumorous lesions in the duodenum. These lesions have been associated with clinical symptoms including diarrhea, obstruction, dyspepsia, ulceration, and gastrointestinal bleeding. We present a case of gastric heterotopia that is unique because the lesions occurred as multiple, carpet-like, nonpolypoid areas throughout a large portion of the small intestine. A review of the literature is included.

Cystic degeneration of heterotopic pancreas.

The case of a large cyst arising from heterotopically-situated pancreatic tissue in an 11-month-old girl is reported. This is the first published report of childhood pancreatic cyst that developed in heterotopic pancreatic tissue. There is strong evidence to suggest that the cyst became symptomatic as a result of secondary infection, an additional unreported phenomenon. This case serves to underscore the fact that pancreatic cysts should be considered in the differential diagnosis of intestinal duplication cysts of childhood.

Stromal microenvironment of human fetal hematopoiesis: in vitro morphologic studies.

The stromal microenvironment is essential for the proliferation and differentiation of hematopoietic progenitors. The features of stroma which contribute to normal hematopoietic stem cell ontogeny or to observed differences in hematopoiesis between fetal and adult hematopoietic organs remain to be fully characterized. In this study, we used long-term culture conditions to grow human fetal liver, fetal bone marrow and adult bone marrow-derived stroma. The stromal layers in all cultures were observed to support multilineage hematopoiesis. Routine and electron microscopic evaluation of the stromal layers reveal the presence of two distinct cell types: a large cell with extensive cytoplasmic projections, and a smaller cell resembling a macrophage. In contrast to some previous reports from in vitro stromal studies, there were no adipocytes, endothelial cells, or cells which could conclusively be identified as fibroblasts in the stromal layer. These findings were further substantiated by negative findings on sections stained with oil-red-O for fat, trichrome for collagen, and factor VIII-related antigen for endothelial elements. There were no morphological differences in the stromal layers from fetal liver, fetal bone marrow, and adult bone marrow sources. This finding is important because it suggests that differences in the behavior of hematopoietic stem cells, when supported in these various hematopoietic microenvironments, are less likely to be explained by obvious differences in the cytologic architecture of stroma than by differences in stem cell biology or growth factor interactions.

Molecular cloning of mouse insulin-like growth factor binding protein 4 (IGFBP4) cDNA and expression of a fusion protein with IGF-binding activity.

The TC-1 bone marrow stromal cell line expresses a 2.3 kb IGFBP-4 mRNA transcript. Reverse transcription/polymerase chain reaction was used to amplify the complete open reading frame of the insulin-like growth factor binding protein-4 (IGFBP-4) from poly(A)+ of a murine bone marrow stromal cell line (TC-1). Sequence analysis reveals that the murine IGFBP-4 is highly homologous to the rat IGFBP-4 and less so to the human IGFBP-4. The inferred amino acid sequence has a molecular weight of 25.7 kD. An IGFBP-4/maltose binding protein fusion peptide expression in the pMal-p2 vector produced a fusion protein exhibiting both IGFBP immunoreactivity and IGF-I binding activity with specificity characteristic of IGFBPs.

Outcome of nonoperative management of splenic injury with nuclear scanning. Clinical significance of persistent abnormalities.

Uncertainties remain about the frequency and need for diagnostic imaging following recovery from splenic injury with nonoperative management. To gain further understanding, the final appearance of the splenic roentgenographic image was evaluated in 20 consecutive children (mean age, 10.1 years) undergoing serial studies up to 70 weeks following injury. A total of 65 technetium 99m sulfur colloid scans, including 45 follow-up studies, were obtained and evaluated. By 20 weeks following injury, six patients (30%) were normal, four (20%) demonstrated minimal residual effects, and 10 (50%) had significantly improved, leaving some persistent abnormality. None of the patients in the last group showed any clinical problem. No distinctions could be made by comparing the severity of the initial injury with a persisting imaging defect. We conclude that clinical considerations alone should determine whether any follow-up imaging be performed in children recovering from splenic injury.

Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.

We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment.

Cutaneous ciliated cyst with interspersed apocrine features presenting as a pilonidal cyst in a child.

We present a case of a cutaneous ciliated cyst that presented in a 13-year-old female. The lesion was felt to be a pilonidal cyst, based on its location and clinical appearance. This case is unusual because it is the youngest reported example of this very rare lesion. In addition, the admixture of apocrine and ciliated cells is extremely unusual. The histogenesis of cutaneous ciliated cysts is considered.