HISTOCHEMICAL STUDIES ON ACHD MUCOPOLYSACCHARIDES

Some new methods for demonstration and differentiation of acid mucopolysaccharides were described.

• 1) 

  Cationic surfactive resin-azo dye method could be used for all kinds of acid mucopolysaccharide demonstration.
• 2) 

  Neutral red method was found to differentiate sulfated and non-sulfated groups on one section.
• 3) 

  Two principles to identify keratosulfate in mesenchymal tissues were studied;

• (1) 

  Hyaluronidase-methylation-saponification method can differentiate keratosulfate B which are both sensitive to testicular hyaluronidase.
• (2) 

  Sugar reactions: Molisch reaction, carbazol-sulfuric acid reaction, etc. can demonstrate the presence of galactose-containing keratosulfate in the frozen sections.

By using these together with well established methods a differential table was made.
The author is deeply grateful to Dr. SHIDA and Dr. KUSHIDA of the General Institute for Medical Biochemistry, Kyoto, Kowa Co., Kaken Yakukako Co., and Hodogaya Chemical Industry Co. for the supply of several kinds of samples.     

Cohen syndrome: the clinical symptoms and stigmata at a young age

We present the clinical findings and follow-up data of four female children with Cohen syndrome, two sisters and one pair of dizygotic female twins. The most characteristic findings from birth on were as follows:

• 1. 

  Low-normal growth parameters at birth.
• 2. 

  Mild hypotonia and evidence of progressive microcephaly with narrow forehead in the first year of life.
• 3. 

  Neutropenia was present from the beginning, remained unchanged over the years and is not associated with higher susceptibility to infections.
• 4. 

  Autistic behavior and severe psychomotor retardation up to the age of 2 years. At that age the ocular anomalies with high-grade myopia and chorioretinal dystrophy were diagnosed. Correction of the myopia resulted in a marked catch-up in psychomotor development.
• 5. 

  After the age of 6 years facial stigmata became more evident with short philtrum of the upper lip and broad and large upper incisors.
• 6. 

  Tendency to truncular obesity with rest hypotonia and poor muscle development after the ages of 6 to 8 years.

The clinical findings and follow-up data in the present four children with Cohen syndrome illustrate that the diagnosis of Cohen syndrome in infancy is very difficult.     

β - 2 - Microglobulin. Part of the HL-A Molecule in the Cell Membrane

Human lymphocytes were reacted with antisera against β-2-microglobulin (β-2-m) or HL-A antigens, and with xenogeneic anti-lymphocyte sera under conditions where redistribution and aggregation of the corresponding cell-membrane components were induced. The results demonstrate that:

• 1) 

  Treatment with anti-β-2-m antisera aggregated and completely removed the HL-A anti-genic structures from the cell membrane, but not, however, the structures reactive with anti-lymphocyte sera.
• 1) 

  Treatment with anti-HL-A antisera also aggregated some of the β-2-m carrying structures, but left others undisturbed.
• 1) 

  Treatment with anti-lymphocyte sera did not disturb expression of HL-A antigens or β-2-m structures.
• 1) 

  Anti-β-2-m and anti-HL-A antisera inhibited antigen activation of lymphocytes, but this was not so in the case of the anti-lymphocyte sera used.
• 1) 

  The anti-β-2-m antiserum and the anti-lymphocyte sera were mitogenic for normal lymphocytes.

Taken together, our results strongly suggest that β-2-m is also part of native cell-membrane bound HL-A antigens, and that the antigenic structures which react with xenogeneic anti-lymphocyte sera may reside on molecules separate from those carrying HL-A and β-2-m activity. Furthermore, the results indicate that the two sub-unit molecules consisting of HL-A alloantigenic determinants and β-2-m are closely associated — structurally or functionally — with lymphocyte receptor structures.     

Natural selection associated with birth weight I. Selection intensity and selective deaths from birth to one month of life

Differential mortality as a function of birth weight was studied up to the 4th week of life in all single births in Italy in 1974.

• (i) 

  Both selection intensity and selective mortality are much higher with increasing immaturity.
• (ii) 

  For babies born at term or after 8 months of pregnancy selection intensity tends io relax as early as one week after birth, while for those born after 7 months selection is at work for a longer period.
• (iii) 

  Selective mortality, on the other hand, keeps increasing after birth but its relevance is relatively decreasing since average mortality after birth continues to decrease.

     

The atopy trait in hypersensitivity to nonsteroidal anti-inflammatory drugs

The prevalence of atopy was evaluated in two groups of subjects with hypersensitivity to nonsteroidal anti-inflammatory drugs (NSAID):

• 1)

  78 patients with aspirin-induced asthma (AIA) confirmed by oral or bronchial provocation challenges
• 2)

  42 subjects with hypersensitivity to pyrazolone drugs (case history and positive skin tests to noramidopyrine/aminophenazone) who tolerated aspirin well.

Fifty sex- and age-matched persons from an unselected general population, with no hypersensitivity to NSAID, formed the control group. Atopy was estimated from the results of the following clinical and biologic parameters:

• 1)

  personal and family history of atopic diseases
• 2)

  skin prick tests with 16 aeroallergens
• 3)

  serum levels of specific IgE to five aeroallergens
• 4)

  total serum IgE level.

Different definitions of atopy were used, consisting of constellations of two or three of the above-mentioned features. The results of the study revealed that the prevalence of atopy varied according to the criteria used for its definition. Irrespective of the definition used, a similar distribution of atopy was observed in both groups of patients with hypersensitivity to NSAID. Atopy was more frequent in either group of patients with intolerance of NSAID than in the control group. Thus, atopy is related to adverse drug reactions to NSAID.     

Cytogenetic studies on cultured fibroblast-like cells derived from basal cell carcinoma tissue

Chromosomal investigations were performed on fibroblast cultures established from tumour tissue of six patients with multiple basal cell carcinoma, and from one patient with a solitary basal cell carcinoma. In four instances, fibroblast cultures from specimens of unaffected skin areas Were examined simultaneously. Metaphases of peripheral blood lymphocytes were analysed in all patients. Three individuals showed increased rates of chromosomal breakage and rearrangement; the possibility of a relationship between these findings and the Occurrence of multiple basal cell carcinoma is discussed:

• 1). 

  The chromosomal aberrations noted in one patient with multiple arsenic-induced basal cell carcinoma probably reflect the long-term effect of exposure to arsenic.
• 2). 

  In the second case, the aberrations found in cultures from unaffected skin and blood lymphocytes may be due to repeated X-ray therapy of multiple nevoid basal cell carcinoma
• 3). 

  In the third patient, likewise affected by multiple nevoid basal cell carcinoma, the etiology of the increased frequency of chromosomally altered cells remains obscure. Taken together with two other observations (Happle et al. 1971, Happle & Kupferschmid 1972), the aberrations could indicate that in some patients with nevoid basal cell carcinoma syndrome the incidence of spontaneous chromosomal breakage tends to increase.

     

Age at symptom onset and distribution by sex and symptoms in patients sensitized to different allergens

The aim of our study was to analyze the clinical features, particularly the age at symptom onset, of allergic subjects (asthma and/or rhinitis) on the basis of the etiologic elements (sensitization to various allergens). We identified a group of monosensitized patients and a group of polysensitized patients. Within these groups, we identified subgroups of subjects monosensitized to one of the five main allergenic mixes (mites, Gramineae, trees, Parietaria, and Artemisia) and five subgroups of patients sensitized nonexclusively, that is, polysensitized, to the same allergens. The comparison between the two groups and among the various subgroups enabled us to conclude that:

• 1)

  mono- and polysensitized patients present some clinical features so different as to constitute two clearly distinct clinical groups
• 2)

  analysis of the clinical features associated with the sensitization to a specific allergen brings us to significantly different conclusions when we consider subgroups of monosensitized or polysensitized patients
• 3)

  the parameter "age at symptom onset" shows great heterogeneity among both the mono- and the polysensitized subgroups - in particular, the great differences in mean age among the monosensitized subgroups (trees>y4rtemi.s(fl>Pflrie/flria>Gramineae>mites) appear very interesting and are open to various interpretative hypotheses
• 4)

  unlike the polysensitized group, in the monosensitized group and subgroups, mean age is similar between men and women and, only for tree- and parietaria -monosensitive patients, also between asthmatic and rhinitic subjects.

     

Electron Microscopic and Histochemical Studies on Vacuolar Alteration of Proximal Convoluted Tubules of Rat Kidney after Hypertonic Sucrose Administration

Changes of the proximal convoluted tubular epithelium of the rats which were sacrificed instantaneously at 5, 15, 30 minutes, 1, 3, 6, and 8 hours after intraperitoneal injections of 40 to 52 cc per kg of a 50 % aquenous solution of sucrose, were observed by electron microscopic and histochemical methods. The results obtained can be summarized as follows:

• 1) 

  The vacuoles begin to appear 15 minutes after the injection in the apical portion of the tubular cytoplasm and become distinct in 30 minutes. After 6 hours, it is observed in the entire cytoplasm which is filled with large vacuoles.
• 2) 

  The vacuoles originate from dilatation of simple pinocytotic vesicles which exist physiologically beneath the brush border microvilli. The small vacuoles of 1 to 1.5 microns in diameter reveal heavy acid phosphatase activity and have temporarily a character of so-called lysosome or cytosome. With the enlargement of vacuoles, the surrounding single limiting membrane reveals discontinuity or rupture at numerous sites. At this stage, the vacuoles aggregate and fuse with each other to form larger ones of 4 to 4.5 microns in diameter and lose their acid phosphatase activity.
• 3) 

  Vacuolar alteration of the proximal convoluted tubules and the role of lysosome and FCD are discussed.

     

Crucial function of the pre-T-cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T-cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lyinpbocyte development:

• *

  The pre-TCR is by far the most effective receptor that generates large numbers of CD4⁺8⁺ T cells with productive TCRβ rearrangements.
• *

  In the absence of the pre-TCR, TCRβ rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCRβ gene.
• *

  The pre-TCR directs developing T cells to the αβ lineage because y5 T cells from pTα^-/- mice proceed much further in TCRβ rearrangement than γδ T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic αβ lineage commitment.

     

Effects of various vacuum cleaners on the airborne content of major cat allergen (Fel d 1)

Background It has been shown that a vacuum cleaner (VC) can increase airborne cat allergen levels. This study aimed to compare the degree of leakage of airborne Fel d 1 levels among five different VCs, both under laboratory conditions and in an apartment with cats.
Methods Three of the VCs were marketed as antiallergic: a HEPA filter VC (VC A), a water impingement and HEPA filter VC (VC B), and a foam fabric filter VC (VC C). The other two were standard VCs: VC D and VC E. VCs were tested in a 20 m', airtight, experimental room and in a 53 m²* living room in an apartment with three cats. Air was sampled with a glass-fiber filter and an impinger at 20 1/min for 30 min before, during, and after vacuuming. Airborne Fel d 1 was measured with a two-site monoclonal ELISA assay.
Results In the experimental room, no airborne Fel d 1 level was measured before using the VCs. After introducing a dust sample containing Fel d 1 in the VCs. we found that VCs A, B, and E did not provoke any increase in airborne Fel d I. In contrast, VCs C and D significantly increased airborne Fel d 1 levels (GM: 4.9 and 5.3 ng/m, respectively). In the apartment, all VCs induced an increase in airborne Fel d 1, which was carried by particles greater than 5 nm. However, VCs C and D provoked significantly greater increases in airborne Fel d 1 than VCs A, B, and E (P=0.0001).
Conclusions Our results suggest that:

• 1)

  The two VCs with leakage in the experimental room had greater leakages in the apartment.
• 2)

  In the apartment with cats, all VCs provoked increases in airborne Fel d 1, primarily carried by large particles.
• 2)

  Given the increased marketing of "antiallergic" VCs, further studies are needed to standardize methods for testing airborne allergen leakage by VCs.

     

On the dynamics of HLA-A2 antigen-antibody complexes on the cell membrane of human peripheral blood lymphocytes. A biochemical and electron microscopical autoradiographic study

This report deals with some dynamic aspects of membrane bound immune complexes of HLA antigens and antibodies. Radiolabelled HLA antibodies were used to sensitize peripheral blood lymphocytes. Upon in vitro incubation of these cells, two different events were detected:

• 1) Immune complexes and a limited amount of free antibody were shed into the culture supernate; from the immune complexes specific antibodies could be regenerated.

• 2) Immune complexes were also internalized by means of endocytosis as visualized by electron microscopical autoradiography. Antibodies were found above dense bodies (lysosomes) and above multivesicular bodies.

The results are discussed in relation to the biology of membrane bound HLA molecules.     

Cellular damage and prevention in childhood hydrocephalus

The literature concerning brain damage due to hydrocephalus, especially in children and animal models, is reviewed. The following conclusions are reached:

• 1. 

  Hydrocephalus has a deleterious effect on brain that is dependent on magnitude and duration of ventriculomegaly and modified by the age of onset.

       
  
  

Association of HLA-Bw65 with two major complotypes

The association between the HLA-B14 subtypes Bw64 and Bw65 and complement allotypes (C2, Bf and C4) was investigated in both population and family studies. Bf, C4A and C4B allotyping was performed on 37 Bw64 and 35 Bw65 positive unrelated Welsh/English subjects. Sixteen HLA-Bw65 bearing haplotypes were characterized for HLA-ABC, DR and DQ antigens and complement allotypes, including C2. The findings of the population study suggested that the complement haplotype associated with Bw64 is BfS, C4A2, C4B2. The population and family studies revealed two major complement haplotypes associated with HLA-Bw65: (i) C2C, BfF, C4A3, C4A1 - often associated with HLA-A3, Cw8 and DRw13, and (ii) C2C, BfS, C4A2, C4B2 - often associated with HLA-Aw33, Cw8 and DR1 or with A28, Cw8 and DRw13. The HLA-Bw65 bearing haplotypes of three families carried a C4B2B1 duplication of the C4B locus. In these families three C4B gene products were identified in the Bw65 positive members using an anti-C4B monoclonal antibody. It is suggested that most, if not all, HLA-Bw65 bearing haplotypes may possess a C4B locus duplication.     

Idiopathic hemochromatosis. Immunogenetics and diagnosis. Prevention by HLA genotypes

Idiopathic hemochromatosis is an hereditary iron overload. The study of HLA types demonstrated clearly the linkage with HLA system. The preferential correlation established with A3 (72%) but other alleles were linked: B7, B14. HLA alleles were only the markers of hemochromatosis allele (H) and were not implicated in other iron overload. Family studied, defined two linked haplotypes: A3, Cw7, B7, Bw6, BfS, DR2, GLO1 et A3, Cw8, B14, Cw6, BfF, DRw6, GLO2. Demonstration of the recessive mode of inheritance was established by family studies. The affected siblings had the same HLA haplotype that the proband and homozygous or heterozygous expressed state was assessed in siblings. The HLA family types allowed to detect in 147 families 88 potential diseased patients among of them 73% had early blood-drawing.     

MS strategies for high throughput glycomics and glyco-proteomics

Ultra-high sensitivity mass spectrometric strategies for defining the primary structures of highly complex mixtures of glycopolymers are revolutionizing structural glycobiology in the postgenomic era. MS strategies incorporating MALDI-MS and ES-MS/MS enable very complex mixtures from biological extracts and/or glycopolymer digests to be screened, thereby revealing the types of glycans present and, importantly, providing clues to structures that are likely to be functionally important.
This lecture will overview strategies employed in our laboratory which enable the glycome of cells, tissues and organs to be examined and the glycoforms of individual glycoproteins to be identified. The types of structural problems that can be solved with current technology will be exemplified by some of our recent research in the following areas:

• • 

  Development and application of glycomics screening strategies for examining glycosylation changes in cells and tissues of knockout mice.

       
  
  

The HLA system in the Korean population

The frequencies of HLA-A, B, C, DR, and DQ antigens, HLA-D (HTC-defined) haplotypes, and the HLA-linked genetic markers glyoxalase I (GLO), factor B (Bf), C2 and C4 were studied in 162 healthy unrelated Koreans. Antigens A2, A24, A26, B44, B51, Bw62, B35, Cw1, Cw3, DR2, DR4, DRw6, DR7, and DRw8 were observed at frequencies of 15% or greater, and GLO-2, BfS, C4A*3, C2C, C4A*4, C4B*1, and C4B*2 were also frequently observed. The antigens A23, A25, B18, Bw42, Bw47, and B21 were not observed at all. HLA-DR4 was the most common class II antigen and was associated with a series of HLA-D-defined haplotypes including Dw4, Dw10, Dw13, and Dw15. The HLA-DRw6, DR2,Dw8, and DRw8 haplotypes were also found frequently. DR2 haplotypes were either Dw2 or Dw12, while all DRw8 haplotypes tested corresponded to the DB7 or Dw "8.3" specificity that has been described in other Oriental populations. Significant linkage disequilibrium was found between the alleles A2,Cw1; A30,B13; A30,Cw6; A30,DR7; Cw1,Bw22; Cw5,B12; Cw6,B13; Cw6,DR7; B7,DR1; B12,Dw6; B12,DR7; B12,Dw7; B13,DR7, B17,DR3; Bw22,C4B*6; DRw6,BfF; and C4A*4,C4B*2. A comparison of gene frequencies and commonly observed haplotypes between Koreans, Chinese, Japanese, and Caucasians showed that while Koreans share several characteristics in common with other Oriental populations, there are allelic frequencies and haplotypes in Koreans that are distinct.     

Marked shortage of C4B DNA polymorphism among insulin-dependent diabetic patients

TaqI, BamHI and HinddIII polymorphisms of the C4 genes were studied with a 500-bp C4 cDNA probe (pAT-A153) specific for the 5' end of the gene. The restriction patterns obtained were correlated with the C4A and C4B genotypes in 35 patients suffering from insulin-dependent diabetes mellitus (IDDM), and results were compared to those from 40 healthy individuals. The controls, all Caucasian, were genotyped for HLA-A, B, C, DR, Bf, C2 and C4, together with 10 diabetics and their families; haplotypes for the other patients had been deduced using DNA and protein polymorphism, and taking into consideration linkage disequilibrium for neighbouring loci. No significant difference between genotypes at the C4A locus was seen in either population. The C4A gene deletion, associated with a C4B "short" gene (66.7%), was found mainly in the haplotype B8,Cw7,DR3,BfS,C2C, C4AQOB1, and the C4B gene deletion in the haplotype B18,Cw5,DR3,BfF1, C2C,C4A3BQO. When diabetic patients were compared with normal individuals, we observed, at the C4B locus, a decrease in the C4B "long" gene (22% vs. 49% respectively, p less than 0.001). A compensatory increase was observed in patients vs. controls for the frequency of C4BQO, both in the deleted and intact form (26% vs. 10% respectively, p less than 0.03).     

HLA—A, B, Bf Three Point Association of 1,072 Haplotypes in a German Population

Altogether 1,072 HLA-A, B, Bf haplotypes in 536 parents of 268 German families were analyzed by gene and haplotype counting in order to investigate the two and three point association of HLA-A and B antigens with Bf alleles. In agreement with previously published data (Albert et al. 1975, Bender et al. 1977) significant positive linkage disequilibria were found for HLA-A3 and BfF, B7 and BfS, B8 and BfS, Bw35 and BfF, whereas significant negative Delta values appeared to exist for HLA-A3 and BfS, B7 and BfF, B8 and BfF, B12 and BfS, as well as Bw35 and BfS. Significant positive three point Delta values were found for the following haplotypes: HLA-A11, Bw35, BfS, HLA-Aw23, Bw35, BfS, HLA-A28, B12, BfS, HLA-A2, B17, BF, HLA-A3, Bw35, BfF, HLA-Aw24, b12, BfF and HLA-A29, B12, BfF. According to the large number of comparisons performed calculating the level of significance of the three point associations, these data have to be corroborated by further investigation. The three point Delta values, obtained by calculation from the patients' phenotypes differed markedly from those results obtained by haplotype counting.     

Studies on an Isolated West Indies Population: I. Analysis of HLA Genotypes

The transmission of HLA genes was studied in an isolated population of French origin on the lesser Antilles islands in the West Indies. The study of 74 unrelated individuals, 44 of whom were genotyped, was carried out for the alleles of HLA loci: A, B, C and Bf (proactivator factor of properdin).
As a result of the founder effect and the inbreeding process, the HLA haplotypcs were noted to be less polymorphic than in a French continental population. Two haplotypes: A2, Cw5, B12, BfS and A3, C-, B14, BfF represent 24% of the observed haplotypes, and only 2% of the reference haplotypes in France.
No significant excess or deficit of homozygotes was observed at the A and B loci.