Identification of a single MPV17 nonsense-associated altered splice variant in 24 South African infants with mitochondrial neurohepatopathy.

MPV17-related mitochondrial neurohepatopathy is a rare genetic disorder worldwide. We report on a novel pathogenic variant in the MPV17 gene in 24 unrelated neurohepatopathic infants of non-consanguineous Black South African heritage. Exome sequencing identified homozygosity for a c.106C>T nonsense variant in exon 3 of the human MPV17 gene in 2 unrelated index patients. mRNA analysis revealed transcripts both with and without exon 3, indicating both reduced splice efficiency and premature termination as mechanisms for disease. Carrier frequency in this population was found to be 1 in 68 (95% CI; 1/122-1/38) with an estimated newborn incidence of 1 in 18 496 (95% CI; 1/59 536-1/5776). Affected infants all presented with infantile onset neurohepatopathy with none surviving beyond infancy. This description of a relatively common pathogenic variant underlying a previously uncharacterized severe neurohepatopathy in South Africa will engender increased awareness, earlier diagnosis and possibly improve outcome if preventative or specific therapeutic options can be found.

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix.

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.

Platelet-rich plasma sequestration, with therapeutic platelet yields, reduces allogeneic transfusion in complex cardiac surgery.

UNLABELLED: Platelet dysfunction is the most common cause of nonsurgical bleeding after cardiopulmonary bypass (CPB). We hypothesized that reinfusion of a therapeutic quantity of platelets sequestered before CPB would decrease the need for allogeneic platelet transfusion, as well as decrease bleeding and total allogeneic transfusion, in cardiac surgery patients at moderately high risk for bleeding. Fifty-five patients undergoing either reoperative coronary artery bypass (CABG) or combined CABG and valve replacement were randomized to control or platelet-rich plasma sequestration (pheresis) groups. All patients received intraoperative epsilon-aminocaproic acid infusions. There was no significant difference between groups with respect to preoperative characteristics, duration of CPB, or target postoperative hematocrit. Mean platelet yields were 6.2 +/- 2.1 units (3.1 x 10(11) platelets). Mean pheresis time was 44 min. Allogeneic platelets (range = 6-12 units) were transfused to 28% of control patients, compared with 0% of pheresis patients (P < 0.01). Allogeneic packed red blood cells were transfused to 45% of control patients (1.2 units per patient) versus 31% of pheresis patients (0. 7 unit per patient) (P = 0.35). Total allogeneic units transfused were significantly reduced in the pheresis group (P < 0.02). Mediastinal chest tube drainage was not significantly decreased in the pheresis group. In this prospective, randomized study, therapeutic platelet yields were obtained before CPB. In contrast with recent studies with low platelet yields, these data support the conclusion that platelet-rich plasma sequestration is effective in reducing allogeneic platelet transfusions and total allogeneic units transfused in cardiac surgery patients at moderately high risk for post-CPB coagulopathy and bleeding. IMPLICATIONS: Transfusion of allogeneic blood products, including platelets, is common during complex cardiac surgical procedures. In the present prospective, randomized study, a significant reduction in allogeneic platelet transfusion and total allogeneic units transfused was observed after the reinfusion of a therapeutic quantity of autologous platelets sequestered before cardiopulmonary bypass.

The effect of carbamazepine and sodium valproate on the blood and serum values of children from a third-world environment.

In third-world countries many children with epilepsy also suffer from malnutrition, anemia, liver disease, and immunosuppression. Doctors might have reservations about the use of anticonvulsants that could aggravate these disorders. The purpose of this study was to establish the prevalence of abnormal blood and serum values in children receiving carbamazepine or sodium valproate as monotherapy who attended a child neurology clinic serving a third-world community in Cape Town, South Africa Blood samples were taken at routine follow-up visits from 104 children who had been on carbamazepine or sodium valproate monotherapy for at least 6 months. Hematology, serum chemistry, immunoglobulins, and anticonvulsant levels were measured by standard laboratory procedures. Very few subjects had any values outside accepted normal ranges. When clinically indicated and available, carbamazepine and sodium valproate can be prescribed for children from a third-world environment. Frequent blood and serum testing is not necessary in asymptomatic individuals.

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

Childhood secondary (symptomatic) epilepsy, seizure control, and intellectual handicap in a nontropical region of South Africa.

PURPOSE: To determine the prevalence of secondary (symptomatic) epilepsy and to ascertain levels of seizure control and intellectual function in a clinic population of children drawn from a poor community in a temperate region of South Africa. METHODS: Detailed review of the records of every child with recurrent seizures who attended a special epilepsy outpatient clinic during 1995. RESULTS: A total of 1,017 children was studied. In 432 (43%), there was historic, clinical, and radiologic evidence to suggest that epilepsy was symptomatic of underlying brain damage or defect. Acceptable seizure control was maintained with a single standard anticonvulsant drug (AED) in 65% of cases. In the 6 months preceding the study, 37% of the subjects had remained seizure free. Forty-seven percent of the study population were considered to be of subnormal intelligence. CONCLUSIONS: In a children's hospital outpatient population in the Western Cape region of South Africa, the prevalence of secondary epilepsy is higher than is found in developed countries. There is need within the community for preventive measures aimed at reducing the prevalence of perinatal insults, meningitis, tuberculosis, neurocysticercosis, and cerebral trauma.

A comparison of the effect of growth factors on intestinal function and structure in short bowel syndrome.

BACKGROUND/PURPOSE: Epidermal growth factor (EGF) and Insulin like growth factor-1 (IGF-1) increase substrate absorption beyond the normal adaptive response after massive small bowel resection in the rat. However, the mechanism for this response is unknown. This study was designed to evaluate the ultrastructural features of the rat small intestine epithelium after exposure to EGF and IGF-1 and correlate any changes with a possible hypothesis regarding the mechanism for the increased absorption. METHODS: Male Sprague-Dawley rats underwent an 80% small bowel resection and jejunostomy tube placement. Seven days later an osmotic pump placed subcutaneously and containing the test substance was connected to the jejunostomy tube. The rats were assigned to one of three groups: group 1 received normal saline (control, n = 5); group 2 received EGF at 150 microg/kg/d (n = 5); and group 3 received IGF-1 at 20 mg/kg/d (n = 5). After a 14-day infusion, a portion of mid-small bowel was resected for light and electron microscopic evaluation from each of the animals. The following features were compared between the groups: villous length, crypt length, villous-crypt ratio, villi per millimeter mucosa, goblet cell distribution, eosinophilic infiltrates, number and distribution of organelles, length of microvilli, and completeness of microvillous surface. RESULTS: Ultrastructurally, the bowel epithelium was well preserved in all animals. There were no objective ultrastructural differences between the controls and growth factor-exposed animals. The mean villous-crypt ratio, mean number of villi per millimeter of mucosa (cross section), and mean microvillous height were not significantly different among the groups. However, there was a subjective increase in the number of lysosomes in the enterocytes exposed to EGF and IGF-1. CONCLUSIONS: Administration of EGF and IGF-1 after massive small bowel resection does not appear to significantly alter the small intestine epithelial ultrastructure when compared with the control group. The increase in lysosomes in some of the enterocytes of the animals exposed to growth factors may be important because this finding was not seen in any of the control electron photomicrographs. Studies to evaluate enterocyte gene and protein expression are necessary to determine the mechanism of EGF and IGF-1 enhancement of substrate absorption beyond intestinal adaptation.

Estrogen and progesterone receptors in salivary gland adenoid cystic carcinoma.

Adenoid cystic carcinomas of salivary glands occur more frequently in women and bear remarkable similarity to adenoid cystic carcinomas of the breast. In addition, breast carcinomas express estrogen and progesterone receptors that impact prognostic significance. This suggests a possible role for sex steroid hormones in the development and progression of salivary gland adenoid cystic carcinoma. On this basis, 12 samples of formalin-fixed, paraffin-embedded salivary gland adenoid cystic carcinomas and 12 samples of normal salivary gland tissue were immunohistochemically evaluated for estrogen and progesterone receptor protein expression. Estrogen receptors were not detected in either group; however, a significantly higher progesterone receptor level was evident in the neoplastic group compared with normal tissue (p < 0.01). These data confirm the presence of progesterone receptors within normal and neoplastic salivary gland tissue. Progesterone receptor expression may be of possible prognostic and therapeutic value in some cases of adenoid cystic carcinoma.

Estrogen receptor detection in paraffin sections of adenocarcinoma of the colon, pancreas, and lung.

We performed a retrospective analysis for the presence of estrogen receptors (ERs) in known cases of adenocarcinoma of the colon, pancreas, and lung. Estrogen receptor status was determined by immunohistochemical methods using the ER-ICA (Abbott Laboratories, North Chicago, Ill) antibody on paraffin sections. None of 43 colon and 18 pancreatic adenocarcinomas demonstrated ERs by this method. Three of 42 lung adenocarcinomas exhibited some degree of nuclear staining for ERs. When evaluating an adenocarcinoma of unknown primary site, the ER-ICA antibody can be useful when included in a panel of antibodies. Positive results tend to exclude colonic or pancreatic, but not pulmonary, primary sites.

Diagnosis of oral hairy leukoplakia by ultrastructural examination of exfoliative cytologic specimens.

Lingual exfoliative cytologic specimens (scrapings) were obtained from 18 patients positive for human immunodeficiency virus with clinical oral hairy leukoplakia. Buccal mucosal scrapings were obtained from 12 of these patients. The specimens were processed for examination by transmission electron microscopy (TEM). Sixteen (89%) of the lingual specimens revealed infection of keratinocytes by herpes-type virus. There was no evidence of virus infection in the 12 buccal mucosal scrapings. Fungal hyphae were seen by TEM in 14 (78%) of the lingual scrapings and two (17%) of the buccal scrapings. One exfoliative specimen and two biopsy specimens were stained for Epstein-Barr virus DNA with a DNA probe. The demonstration of herpes-type virions by TEM in keratinocytes from a lesion clinically suspected to be hairy leukoplakia provides direct, objective diagnosis. Furthermore, use of exfoliative cytologic specimens provides a clinically simple, noninvasive technique.

Cytomegalovirus-infected cells express Leu-M1 antigen. A potential source of diagnostic error.

The authors examined cytomegalovirus (CMV)-infected tissues and Hodgkin's Disease (HD) cases with immunohistochemical assays for Leu-M1 and CMV. The cytologic characteristics were correlated with immunostaining patterns. Cytomegalovirus-infected cells in lymph node, lung, and esophagus sections showed Cowdry type A inclusions, and many had granular cytoplasmic inclusions. All infected cells showed nuclear staining with an anti-CMV antibody. Leu-M1 reacted with CMV-infected cells in cytoplasmic areas, particularly near the nucleus simulating the characteristic staining pattern of Reed-Sternberg (R-S) cells. Cytoplasmic staining intensified as the intranuclear inclusions increased in size. Reed-Sternberg cells showed characteristic Leu-M1 positivity along the cell membrane and golgi zone. At times, Leu-M1 staining of CMV-infected cells was indistinguishable from that of R-S cells. None of the R-S cells reacted with the antibody to CMV. Recognition of the reactivity of Leu-M1 with CMV-infected cells is important in avoiding misdiagnosis of CMV lymphadenitis as HD.

Variable region primary structures of a high affinity anti-fluorescein immunoglobulin M cryoglobulin exhibiting oxazolone cross-reactivity.

Previous studies of murine IgM hybridoma protein 18-2-3, derived from an (NZB/NZW)F1 mouse following hyperimmunization with fluorescein (Fl)-conjugated keyhole limpet hemocyanin, demonstrated a high affinity for Fl (Ka = 2.9 x 10(10) M-1) and cryoprecipitation that was abrogated upon Fl binding to the antibody-combining site. V region sequences of 18-2-3 were determined by Edman degradation and nucleotide sequence analysis. The VH region of 18-2-3 was encoded by a gene VHI(B) of the Q52 VH family with 96% homology to anti-oxazolone antibody NQ7.5.3 but utilized a larger D region (DQ52 plus N region). The V kappa region of 18-2-3 was encoded by a gene V kappa IV with an amino acid sequence 97% homologous to that of anti-oxazolone antibody NQ11.1.18. Although monoclonal anti-Fl antibodies 18-2-3 and 4-4-20 possessed similar binding affinities and quenched bound fluorescein to the same extent (Qmax greater than 96%), they utilized different VH, D, V kappa, and J kappa genes, but the same JH gene segment (JH4). Solid-phase analyses showed that 18-2-3 was not idiotypically related to 4-4-20 and 9-40, prototypic anti-Fl antibodies. Fine specificity binding patterns of Fl analogues by 18-2-3 IgM and IgMs were distinct from other anti-Fl antibodies. Monoclonal antibody 18-2-3 bound phenyloxazolone bovine serum albumin with a lower affinity than for Fl-bovine serum albumin. The first hypervariable region of the 18-2-3 light chain showed homology to human cryoglobulins. This is the first variable region sequence of a murine IgM which self-aggregates at low temperature.

Comparison of variable region primary structures within an anti-fluorescein idiotype family.

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.

Single-chain antigen-binding proteins.

Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

Myoglobinuric renal failure after generalised tonic-clonic seizures. A case report.

A 47-year-old man developed progressive renal impairment after a series of seven generalised tonic-clonic seizures. The patient did not become oliguric and because recovery of renal function was rapid, dialysis was not required. The diagnosis of myoglobin-induced renal failure was made on the basis of markedly elevated muscle enzyme values, and myoglobin in the urine.

Fine-structural alterations in Leishmania tropica within human macrophages exposed to antileishmanial drugs in vitro.

The mechanism of action of antileishmanial compounds is poorly understood. Ultrastructural changes in Leishmania tropica within human macrophages exposed in vitro to Pentostam, pentamidine, amphotericin B, WR 6026, ketoconazole, and Formycin B were examined in these experiments. In Pentostam-treated cultures, some organisms exhibited diminished definition of mitochondrial and other membranes, while other organisms had completely disintegrated. Pentostam-exposed macrophages demonstrated loss of membrane definition in the absence of further alterations; it is therefore hypothesized that impaired macrophage membrane function may contribute towards the effect of this drug against macrophage-contained organisms. Leishmania parasites in pentamidine-treated cultures initially demonstrated swollen kinetoplasts and fragmentation of the kinetoplast DNA core. The initial observed effect of the other four drugs on the parasites was cytoplasmic condensation. These ultrastructural studies suggest that all five non-antimonial drugs may have different mechanisms of action than antimony (Pentostam) against Leishmania.

The T-axial membrane system in striated muscles of the horseshoe crab.

Cardiac muscle and skeletal muscles powering walking leg, tailspine and gill movements in Limulus polyphemus were studied by transmission electron microscopy. All muscles examined had extensive invaginations of the sarcolemma at the Z disc and at the lateral margins of the A band. Invaginations at the Z disc were often branched in the transverse and longitudinal planes, but branching was not observed at other locations within a sarcomere. Dyads, and occasional triads, were observed at the A band in all muscles examined.