The human P(k) histo-blood group antigen provides protection against HIV-1 infection.

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

The molecular diversity of Sema7A, the semaphorin that carries the JMH blood group antigens.

BACKGROUND: Semaphorin 7A (Sema7A), the protein that carries the JMH blood group antigen, is involved in immune responses and plays an important role in axon growth and guidance. Because previous serologic studies on red blood cells (RBCs) suggested a considerable diversity of Sema7A, the present study was designed to elucidate the complex picture of the molecular diversity of this protein. STUDY DESIGN AND METHODS: The JMH antigen status was determined by serology, flow cytometry, and Western blot. Genomic and transcript analysis of SEMA7A was performed by nucleotide sequencing. Recombinant Sema7A proteins were used for genotype-phenotype correlation. A three-dimensional model of Sema7A was generated for topologic analyses. RESULTS: Our studies on 44 individuals with unusual JMH phenotypes and their family members revealed that aberrant Sema7A expression can be an inherited or an acquired phenomenon and is based on reduced surface expression or qualitative changes in Sema7A. These different phenotypes are caused by variations of the SEMA7A gene or seem to be generated by autoimmune-related or RBC lineage-specific mechanisms. The variant JMH phenotypes were related to the presence of missense mutations in SEMA7A, predicting amino acid changes in the semaphorin domain of Sema7A. Sequence analysis of the variant SEMA7A alleles revealed mutations affecting codons 207 and 460/461. Topologic analyses showed that Sema7A polymorphisms were prominently located on the top and bottom of the semaphorin domain, suggesting a functional relevance of these sites. CONCLUSION: These findings provide a basis with which to delineate the various ligand-binding surfaces of Sema7A.

Identification of six new alleles at the FUT1 and FUT2 loci in ethnically diverse individuals with Bombay and Para-Bombay phenotypes.

BACKGROUND: The Bombay and para-Bombay phenotypes arise from mutations of the FUT1 gene that silence the gene or affect the efficiency of the encoded 2-alpha-fucosyltransferase. Samples from seven individuals of different geographic backgrounds whose red blood cells had an apparent Bombay or para-Bombay phenotype were investigated. Among these, novel FUT1 and FUT2 alleles were identified. STUDY DESIGN AND METHODS: Standard serologic techniques were used. Genomic DNA was sequenced with primers that amplified the coding sequence of FUT1 and the related secretor gene, FUT2. Routine ABO genotyping analysis was performed. RESULTS: Five new FUT1 alleles were identified that silenced FUT1 or weakened alpha2FucT1 activity. These were 35C>T, 269G>T (Ala11Val, Gly89Val); 421A>G (Trp140Stop); 538C>T, 1089T>G (Gln180Stop, Ala363Ala); 689A>C (Gln230Pro); and 917C>T (Thr305Ile). In addition, both homozygosity and heterozygosity for the previously reported mutation, 826C>T (Gln276Stop), were observed. Four of seven samples were homozygous for the silencing mutation 428A in FUT2. One new FUT2 allele was identified: 278C>T, 357C>T (Ala93Val, Asn119Asn). CONCLUSIONS: These results add to the growing database of apparently sporadic and random mutations in the FUT1 gene and confirm previous reports regarding the lack of ethnic bias. In contrast, our data reinforce the apparent maintenance of the common nonsecretor FUT2 alleles in the population.

New and unusual O alleles at the ABO locus are implicated in unexpected blood group phenotypes.

BACKGROUND: In the ABO blood group system mutations in the A gene may lead to weak A subgroups owing to a dysfunctional 3-alpha-N-acetylgalactosaminyltransferase. STUDY DESIGN AND METHODS: Blood and DNA were investigated to correlate weak A phenotypes with genotype, and an overrepresentation of the infrequent O2 allele was observed. Consequently, 57 available O2 alleles were examined in detail. RESULTS: Two new O2 alleles were identified having mutations resulting in Gly229Asp with or without Arg217Cys. A recently described O2 variant (488C>T; Thr163Met) was also found. Surprisingly, both the original and the variant O2 alleles were associated with either O or Aweak phenotypes. Three novel O alleles surfaced in six other samples with suspected A subgroups. These were A1-like alleles having nonsense mutations causing premature truncation at codons 56, 107, or 181. A second example of the rare O3 allele was also identified. A newly described O1 allele having 768C>A was found to be the third most frequent O allele among Swedish donors. Of the five novel O alleles, three were incorrectly interpreted as A1 following routine ABO genotyping. CONCLUSION: Apparent O alleles lacking 261delG may cause weak A expression on red blood cells and/or inhibit anti-A production. A hypothesis that exchange of genetic material between principally dissimilar O alleles during mitosis ("autologous chimerism") restores glycosyltransferase activity in some cells would explain this interesting phenomenon.

CD151, the first member of the tetraspanin (TM4) superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in kidney and skin.

Tetraspanins are thought to facilitate the formation of multiprotein complexes at cell surfaces, but evidence illuminating the biologic importance of this role is sparse. Tetraspanin CD151 forms very stable laminin-binding complexes with integrins alpha3beta1 and alpha6beta1 in kidney and alpha3beta1 and alpha6beta4 in skin. It is encoded by a gene at the same position on chromosome 11p15.5 as the MER2 blood group gene. We show that CD151 expresses the MER2 blood group antigen and is located on erythrocytes. We examined CD151 in 3 MER2-negative patients (2 are sibs) of Indian Jewish origin with end-stage kidney disease. In addition to hereditary nephritis the sibs have sensorineural deafness, pretibial epidermolysis bullosa, and beta-thalassemia minor. The 3 patients are homozygous for a single nucleotide insertion (G383) in exon 5 of CD151, causing a frameshift and premature stop signal at codon 140. The resultant truncated protein would lack its integrin-binding domain. We conclude that CD151 is essential for the proper assembly of the glomerular and tubular basement membrane in kidney, has functional significance in the skin, is probably a component of the inner ear, and could play a role in erythropoiesis.

Additional molecular bases of the clinically important p blood group phenotype.

BACKGROUND: The purpose of this study was to explore the molecular basis of the p phenotype by analysis of the recently cloned 4-alpha-galactosyltransferase gene responsible for synthesis of Pk (Gb3) antigen. STUDY DESIGN AND METHODS: Forty samples from individuals of eight different nationalities were investigated by serologic methods and DNA sequencing of the Pk gene. RESULTS: Ten different Pk-null alleles, of which 6 are novel, were encountered. The 29 Swedes were homozygous for M183K or G187D, with the former as the predominant allele. Three Israelis were homozygous for a single-nucleotide deletion at codon 219 that shifts and truncates the reading frame by 5 amino acids. Two Italians were homozygous for a triplet deletion causing F81del, while an English donor was heterozygous for F81del but also carried another allele with a combined deletion and insertion. A Pole was heterozygous for alleles with either a single-base deletion at codon 257 or a mutation causing S97L. A Norwegian person and a Japanese person were homozygous for single-base insertions causing a premature stop at codon 282 or extension of the protein by 92 residues, respectively. In 2 samples no mutations were detected. CONCLUSION: The genetic heterogeneity underlying the p phenotype is further emphasized by this study. To date, 11 p-specific mutations have been found in 14 distinct alleles.

The rare Lu:-6 phenotype in Israel and the clinical significance of anti-Lu6.

BACKGROUND: Lu6 is a high-incidence antigen of the Lutheran blood group. Examples of anti-Lu6 are rare and are of uncertain clinical significance. CASE REPORT: Three patients were encountered in whom anti-Lu6 was detected on pretransfusion screening. The patients were all Iranian Jews and were not known to be related. In vitro studies to ascertain the potential clinical significance of the antibody using the monocyte monolayer assay (MMA) were negative in two patients. The third patient received a two-unit transfusion of incompatible Lu6 RBCs with no signs of hemolysis. However, after the transfusion, the MMA and a chemiluminescence test were positive, whereas a chromium survival study was normal. Thus, the antibody may have changed in its clinical significance. CONCLUSION: Although anti-Lu6 does not appear to be a clinically significant RBC antibody in all circumstances, transfusion of Lu6 RBCs in patients with anti-Lu6 should be performed cautiously.

The growth of cartilage canals

Investigations into the growth of cartilage canals, in the cartilaginous upper end of the developing tibia in foetal sheep, were conducted along three lines: (1) The blood vessels were injected with visually opaque fluid and the selected cartilage was cleared. As the cartilage increased in size, the side-arms of a T-shaped canal common to all specimens increased their transverse extent. This point to active penetration of the cartilage by the vessels. (2) The vascular pattern within individual canals was examined histologically in serial sections. The tissue at the end of every canal received arterial blood first, perhaps because an active metabolic process (erosive) was taking place there. (3) The tissue between the vessels and the surrounding cartilage was examined histologically and histochemically in serial sections. a) At the end of a canal this tissue was minimal, chondrocyte capsules were breached on the side facing the vessels, chondrocyte glycogen was diminished but not entirely lost close to the vessels and some glycogen was present within the canal, multinucleate chondroclasis with high ribonucleic acid content were also present within the canal. b) Elsewhere along the canal the intervening tissue was greater in amount, glycogen was absent from the chondrocytes close to the vessels and also absent from the interior of the canal, cytoplasmic ribonucleic acid was not increased or near the canal, multinucleate cells were absent. It was concluded that a slow erosize process occurred over the greater part of the canals and produced a gradual increase in its diameters. At the ends of the canals a rapid erosive process contributed to elongation of the canals (AU)

The secondary cuboid in Jamaican feet

Of 54 pairs of feet routinely dissected over a period of ten consecutive years, two pairs showed the features of "secondary cuboid fused to the cuboid: these are described in detail. The evidence for the existence of an independent secondary cuboid is critically discussed, the existence of this element is not proven. It is suggested that the skeletal variations involving the adjacent parts of calcaneus, navicular and cuboid, including the accessory bone(s) in the interspace between them, are dependent on two factors: (1) absence of rigidly determined bounderies between these three constant bones, and (2) interference with the normal migration of the cuboid during the latter half of the second foetal month. The same factors may be responsible for the formation of calcaneo-navicular bar, which is frequently present in association with peroneal spastic flat foot (AU)

The patterns of cartilage canals

The pattern of vascular canals in the cartilagenous upper end of the developing tibia was investigated in the sheep, goat, rabbit, cat, rat and man. The blood vessels were injected with a visually opaque fluid, and the cartilage was fixed, dehydrated and cleared. Specimens immersed in their clearing fluid were examined visually in transmitted light with a stereoscopic microscope. In each species the upper tibial cartilage showed a constant vascular canal pattern at all stages prior to development of the secondary centre of ossification. The pattern was constant in broad outline only, and allowed great variation in individual details. The canal pattern in any one species differed from the patterns of all other species studied. The different patterns were not obviously related to differences in the size and/or shape of the homologous cartilage in the different species examined. The course and distribution of the vascular canals indicated that the presence of these canals in cartilage cannot be explained soley as the result of passive inclusion of perichondrial cartilage around them (Summary)

Serogical and genetical observations on the blood group A3 B

Two family studies have been performed where the prositus in each case was A3B. In neither cases could this grouping be substantiated on the grounds of genetical inheritance of the gene A3. It is considered in both families that the examples of apparent A3B are really examples of A2B, where there is evidence of gene interaction. The reasons for not accepting these cases as true examples of A3B are discussed (Summary)

Critical staining of pancreatic alpha granules with phosphotungstic acid hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3 percent KMnO4 acidified with 0.3 percent (w/v) H2SO4, decolourised in 4 percent potassium metabisulphite, mordanted 20 min to 2 hr in 4 percent iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95 percent ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended (AU)