Defence context for the UK's Defence Engagement (Health).

Defence Engagement (DE) has been a core UK Defence task since 2015. DE (Health) is the use of military medical capabilities to achieve DE effects within the health sector to achieve security and defence objectives. DE (Health) practitioners must understand the underlying defence context that shapes these objectives. The strategic context is becoming more uncertain with the return of great power competition layered on enduring threats from non-state actors and transnational challenges. The UK response has been to develop the Integrated Review, outlining four national security and international policy objectives. UK Defence has responded by developing the integrated operating concept, differentiating military activity between operating and warfighting. Engage is one of the three functions of operate activity, which is complementary to the other operate functions of protect and constrain. DE (Health) can play a unique role in engagement, given its ability to develop new partnerships through health-related activity. DE (Health) may be an enabler for other engagements or to enable the protect and constrain functions. This will be dependent on delivering improvement in health outcomes. Therefore, the DE (Health) practitioner must be conversant with both the contemporary defence and global health contexts to deliver effective DE (Health) activities. This is an article commissioned for the DE special issue of BMJ Military Health.

Factors that influence the therapeutic activity of 5-fluorouracil [6RS]leucovorin combinations in colon adenocarcinoma xenografts.

The therapeutic activity of FUra alone or combined with [6RS]LV doses ranging from 50 to 1,000 mg/m2 was examined in eight colon adenocarcinoma xenografts, of which five were established from adult neoplasms (HxELC2, HxGC3, HxVRC5, HxHC1, and HxGC3/c1TK-c3 selected for TK deficiency) and three were derived from adolescent tumors (HxSJC3A, HxSJC3B, and HxSJC2). The growth-inhibitory effects of FUra were potentiated by higher doses of [6RS]LV (500-1,000 mg/m2) in three lines (HxGC3/c1TK-c3, HxSJC3A, and HxSJC3B) and by a low dose of [6RS]LV in only one tumor (HxVRC5). Expansion of pools of CH2-H4PteGlun+H4PteGlun (greater than or equal to 2.4-fold) in response to higher doses of [6RS]LV was obtained in all lines except HxHC1. Metabolism of [6RS]LV was high in HxVRC5, with high levels of 5-CH3-H4PteGlu being detected, but not in HxHC1, in which levels of 5-CH3-H4PteGlu and CH = H4PteGlu+10-CHO-H4PteGlu remained relatively low. In the adolescent tumors, levels of CH = H4PteGlu+10-CHO-H4PteGlu were consistently higher than those of 5-CH3-H4PteGlu following [6RS]LV administration, and in HxSJC3A, in which pools of CH2-H4PteGlun+H4PteGlun were significantly expanded, 5-CH3-H4PteGlu concentrations were lower than those observed in the other two lines. The sensitivity of tumors to FUra +/- [6RS]LV and the characteristics of [6S]LV metabolism did not correlate with the activity of CH = H4PteGlu synthetase, the enzyme responsible for the initial cellular metabolism of [6S]LV to CH = H4PteGlu. Thus, no single metabolic phenotype correlated with the [6RS]LV-induced expansion of CH2-H4PteGlun+H4PteGlun pools. Potentiation of the therapeutic efficacy of FUra by [6RS]LV was observed in HxGC3/c1TK-c3 xenografts but not in parent HxGC3 tumors, demonstrating the influence of dThd salvage capability in the response to FUra-[6RS]LV combinations. Plasma dThd concentrations in CBA/CaJ mice were high (1.1 microM). The present data therefore demonstrate the importance of (1) higher doses of [6RS]LV, (2) expansion of pools of CH2-H4PteGlun+H4PteGlun, and (3) dThd salvage capability in potentiation of the therapeutic efficacy of FUra in colon adenocarcinoma xenografts. The plasma levels of FUra achieved in mice are presented.

Development and evaluation of a method to determine indicator microorganisms in air emissions and residue from medical waste incinerators.

To allow testing of microbial destruction in medical waste incinerators, methods were developed to determine indicator microorganisms (Bacillus Stearothermophilus spores) in incinerator air emissions and residue. The emission trapping train consisted of a water cooled glass probe and impingers containing a neutral phosphate buffer. In field tests, spores were injected directly into the probe, and results showed that approximately 60 percent of the spores were recovered. Spores were analyzed with adequate precision using a microbial membrane filter unit. Lab experiments indicated that spores were stable in neutral pH phosphate buffer for up to 20 days, and heat shocking samples (heating to 80 degrees C for 20 minutes) reduced spore numbers in acidic or basic buffer. Laboratory tests also showed that 60 to 70 percent of spores initially added to ash were recovered up to 22 days after addition of the spores. In addition, lab tests showed that spores can be effectively recovered from residue test pipes spiked with indicator spores.

Influence of dose of [6RS]leucovorin on reduced folate pools and 5-fluorouracil-mediated thymidylate synthase inhibition in human colon adenocarcinoma xenografts.

Using preclinical models of human colon adenocarcinomas in immune-deprived mice, the influence of dose of [6RS]leucovorin ([6RS]LV, 20 to 1000 mg/m2) administered by 24-h i.v. infusion was determined on the following parameters: (a) plasma concentrations of the active [6S] and inactive [6R] isomers of [6RS]LV and the biologically active diastereoisomer of 5-methyltetrahydrolate (5-CH3-H4PteGlu); (b) expansion of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), that may influence the binding of 5-fluorodeoxyuridylate to thymidylate synthase; (c) the distribution of polyglutamate forms of CH2-H4PteGlun and H4PteGlun; and (d) (5-fluorouracil (FUra)-mediated thymidylate synthase inhibition in Hx-ELC2, HxGC3, HxVRC5, and HxHC1 tumors. Folypolyglutamate synthetase activities were also determined in each line. Linear increases in plasma concentrations of [6R]LV, [6S]LV, and 5-CH3-H4-PteGlu were determined over the complete range of [6RS]LV doses examined. However, in neoplastic tissues three patterns of biochemical modulation by [6RS]LV were evident. (a) In HxELC2 and HxVRC5 tumors, pools of CH2-H4PteGlun and H4PteGlun were elevated in proportion to the dose of [6RS]LV between dose levels of 50 and 200 mg/m2. Subsequent expansion of these pools continued that was disproportionate to the dose of [6RS]LV until no further increase was observed beyond 800 mg/m2 [6RS]LV, at which point pools were maximally expanded by 4- to 4.5-fold. The extent of retardation of recovery of thymidylate synthase activity increased as the dose of [6RS]LV was increased in both tumors, when FUra (15 or 50 mg/kg), was administered by i.v. bolus injection 3 h into the 24-h infusion of [6RS]LV. This was related to the increase in predominance of CH2-H4PteGlu2-5 with increasing dose of [6RS]LV. (b) For HxHC1 tumors, little expansion of CH2-H4PteGlun and H4PteGlun pools (maximum, 137% of control) was detected at the highest dose levels of [6RS]LV, and no significant modulation of FUra-inhibited thymidylate synthase activity was detected, even at 1000 mg/m2 [6RS] LV. CH2-H4PteGlu5 remained similar or decreased as the dose of [6RS] LV was increased. (c) For line HxGC3, pools of CH2-H4PteGlun and H4PteGlun increased gradually from 169% of control at 20 mg/m2 [6RS] LV to 233% of control at 1000 mg/m2 [6RS]LV, and were intermediate between the expansion observed in HxHC1 in comparison to HxELC2 and HxVRC5 tumors. CH2-H4PteGlu3-5 were elevated at low dose levels of [6RS]LV.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between dose rate of [6RS]Leucovorin administration, plasma concentrations of reduced folates, and pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon adenocarcinoma xenografts.

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H4PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), and the distribution of their polyglutamate species have been examined. Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m2) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H4-PteGlu and 5-methyltetrahydrofolate (5-CH3-H4PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t1/2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC2 and HxGC3 tumors, pools of CH2-H4PteGlun and H4PteGlun were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH2-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin. In HxGC3 tumors, CH2-H4PteGlu4-5 were elevated more rapidly than in line HxELC2, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH2-H4PteGlu5 until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH2-H4PteGlu3-5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-3H]5-fluorodeoxyuridine monophosphate ([6-3H]FdUMP) to thymidylate synthase, and intratumor pools of CH2-H4PteGlun and H4PteGlun were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule for administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the conversion of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon tumors.

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Characterization of the pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in xenografts of human colon adenocarcinoma.

The method for measuring polyglutamate forms of CH2-H4PteGlu and H4PteGlu, by entrapment in ternary complexes with [6-3H]5-fluoro-2'-deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of CH2-H4PteGlu and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relationship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (d) noncovalent complexes formed with PteGlu2-5 could be isolated only at concentrations considerably higher than those required for CH2-H4PteGlu1-6, and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of CH2-H4PteGlu and the combined pools of CH2-H4PteGlu plus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of CH2-H4PteGlu and H4PteGlu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the CH2-H4PteGlu pool, and greater than 93% of the combined pools. A small percentage of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of CH2-H4PteGlu and H4PteGlu was estimated in the range of 0.5 to 2.7 microM in cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 microM. Using thymidylate synthase purified from colon adenocarcinoma HxVRC5, CH2-H4PteGlu5 (where the subscript digit attached to the glutamate portion equals the number of glutamate residues) stabilized the covalent ternary complex at greater than 200-fold lower concentration in comparison to CH2-H4PteGlu1. Data indicated that in each colon tumor, the concentrations of CH2-H4PteGlun or CH2-H4PteGlun plus H4PteGlun were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5-fluorouracil. These data support therapeutic modulation to increase the concentration of CH2-H4PteGlun in the treatment of colon adenocarcinomas with 5-fluorouracil.

Determination of thymidylate synthase activity in colon tumor tissues after treatment with 5-fluorouracil.

The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetrahydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) absorbed greater than 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37 degrees), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.

Relationship between binding affinity, retention and sensitivity of human rhabdomyosarcoma xenografts to Vinca alkaloids.

Xenografts of human rhabdomyosarcoma (RMS) have been derived that differ in their degree of sensitivity to Vinca alkaloids. Lines Rh12 and Rh18 demonstrated, respectively, high and moderate sensitivity to vincristine (VCR), but showed little responsiveness to vinblastine (VLB) in vivo. Rh18/VCR-3, a subline of Rh18 selected for resistance to VCR under in situ conditions, was insensitive to further challenge with VCR. Resistance was associated with elimination of the agent in a biphasic manner, whereas sensitivity to VCR corresponded to very prolonged drug retention in sensitive neoplastic tissues. The initial half-times for drug retention in tumors in vivo (t1/2 alpha) correlated with the degree of sensitivity of tumors to Vinca alkaloids, decreasing t1/2 alpha being associated with decreased sensitivity. A single binding species was observed when membrane-free supernatant fractions were incubated at 37 degrees for 15 min with 10.4 nM [3H]VCR and analyzed by gel filtration HPLC. The protein eluted with a retention time of 57 min and corresponded to a molecular weight (Mr) of approximately 113,000 daltons, agreeing very closely with the Mr of dimeric tubulin (approximately equal to 110,000 daltons). Two fractions were collected and eluted on a one-dimensional denaturing gel. Proteins were transferred subsequently to nitrocellulose and probed with an 125I-labeled monoclonal antibody specific for beta-tubulins. Only the fraction containing bound [3H]VCR contained tubulin. Estimates for the dissociation constants (Kd) for the binding affinity of VCR and VLB in crude, membrane-free supernatant fractions from RMS xenografts were obtained by computer curve fitting using a mathematical binding model. Data fitted a two-site binding model, with Kd values for the high-affinity site ranging from 61 to 160 nM, and for the low-affinity site, from 42 to 94 microM. At physiologically achievable drug concentrations, the relationship between binding affinity, drug retention and tumor sensitivity was examined further. A close relationship was apparent between the Kd values for VCR in Rh12, Rh18 and Rh18/VCR-3 tumor supernatant fractions and VLB in Rh12 preparations, and t1/2 alpha values for drug retention. Prolonged drug retention correlated with a low binding constant. As t1/2 alpha decreased, binding affinity also decreased, as demonstrated by an increase in the Kd value. Consequently, the tightness of drug binding in tumors also correlated with the degree of sensitivity of the xenografts to Vinca alkaloids.(ABSTRACT TRUNCATED AT 400 WORDS)

Relationship between 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthase activity subsequent to 5-fluorouracil administration, in xenografts of human colon adenocarcinomas.

5-Fluorouracil (FUra) has been administered to mice bearing xenografts of human colon adenocarcinomas. In two tumor lines, HxGC3 and HxVRC5, intrinsically resistant to FUra, 2'-deoxyuridylate (dUMP) accumulated 13.4- and 23.9-fold above basal levels. In HxELC2 xenografts, which demonstrated some sensitivity to FUra, there was a decrease in dUMP concentration after drug administration. Maximal intratumor levels of 5-fluoro-2'-deoxyuridylate (FdUMP) were found at 1 hr, but decreased in all tumor lines by 4 hr after administration of FUra. Data derived in tumor cytosols suggested that FdUMP levels in situ were not rate-limiting for formation of covalent ternary complex, but that accumulation of dUMP would retard the rate of complex formation. Subsequent to administration of FUra, thymidylate synthase activity was reduced greater than 75% in all tumors, but it recovered rapidly in tumors resistant to FUra. In addition, the pretreatment level of activity of thymidylate synthase was 12.7-fold greater in HxVRC5 tumors than in HxELC2 tumors. This elevated activity in HxVRC5 tumors appears not to be a consequence of gene amplification. Formation of FdUMP or the accumulation of dUMP did not correlate with the activity of phosphatases measured at pH 5.8 or pH 9.2 in each tumor line. Further, inhibition of phosphatase activity did not alter, significantly, the net rate of dissociation of the FdUMP-thymidylate synthase-[6R]-CH2-H4PteGlu complex.

Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts.

The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2'-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association (ka) for FdUMP was low being between 3.4 +/- 0.9 and 10.2 +/- 2.6 X 10(6) M-1 min-1, with the lowest ka value being determined in cytosols from a tumor (HxELC2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported ka values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH2-H4PteGlu. In cytosols the apparent Km values for dUMP (3.6-4.2 microM) and and [6RS]- CH2-H4PteGlu (25-26.7 microM) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC5 colon adenocarcinoma xenografts. The net dissociation of [6-3H] FdUMP from the covalent ternary complex was 31-33 min in the absence of added CH2-H4PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH2-H4PteGlu required to stabilize ternary complex derived from HxELC2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC3,HxVRC5). Addition of 5-CHO-H4PteGlu, 5-CH3-H4PteGlu, H2PteGlu, and PteGlu did not stabilize the covalent complex, but H4PteGlu substituted for CH2-H4PteGlu.

Microsomal membrane subfractionation by a lectin affinity method.

Concanavalin A-agarose treatment of rat liver post-mitochondrial supernatant removes a fraction rich in cholesterol and 5'-nucleotidase activity but low in glucose-6-phosphatase. At the same time, radiolabel associated with the cell surface is removed. We interpret these findings as evidence that concanavalin A binds to, and under these circumstances will remove, fragments of plasma membrane present in the microsomal fraction and believe that this may be of use in the gentle, and rapid subfractionation of microsomal membranes.

DNA polymorphisms in the apolipoprotein C-III and insulin genes and atherosclerosis.

A total of 167 patients undergoing investigation for suspected coronary artery disease (CAD) were genotyped for restriction fragment length polymorphisms (RFLP) at the apo A-1/C-III locus and the insulin gene locus using cloned human apo A-1 and insulin gene probes. The study group was subdivided into patients with absent or minimal CAD, intermediate CAD and severe obstructive CAD. An Sst-1 polymorphism located in the 3' non-coding region of the apo C-III gene identifies two alleles. One of the alleles (S2) showed a significantly increased frequency in the subjects with severe obstructive CAD (18%) compared with patients with minimal or absent CAD (6%) (P less than 0.025) and normolipidaemic control subjects. This A-1/C-III polymorphism may be a marker for an abnormality in the A-1/C-III genes predisposing to atherosclerosis. In contrast to a previous report, we found no increase in the frequency of the Class 3 insulin alleles in subjects with severe CAD.

Stability of vincristine complexes in cytosols derived from xenografts of human rhabdomyosarcoma and normal tissues of the mouse.

The selective action of vincristine (VCR) has been correlated with longer retention of the drug in neoplastic tissue compared with normal tissues of the mouse (J. A. Houghton, L. G. Williams, P. M. Torrance, and P. J. Houghton, Cancer Res., 44: 582-590, 1984). In order to examine the basis for this differential, the stability of drug-protein complexes was examined further. The stability of drug-protein complexes formed in cytosols derived from HxRh18 tumors, ileum, liver, kidney, skeletal muscle, blood, brain, spleen, lung, and bone marrow was examined. Protein-bound [3H]VCR was isolated by gel filtration of [3H]VCR-cytosol mixtures from each tissue except for ileum and blood. Complexes formed in brain and HxRh18 cytosols were stable at 37 degrees for at least 2 h; all other complexes were unstable. For liver, kidney, and muscle, half-times of complexes were in a similar order to the initial rates of elimination of [3H]VCR from these tissues in vivo but were of shorter duration. The HxRh18-[3H]-VCR complex was unstable at 37 degrees in the presence of cytosols prepared from ileum, kidney, liver, and lung. Drug metabolism by these tissues was not detected in vitro. In the presence of heat-treated extracts from ileum or kidney, [3H]VCR complex was stable, suggesting that the destabilizing factor may be enzymic. Degradation of 125iodinated tubulin, analyzed by polyacrylamide-sodium dodecyl sulfate gel electrophoresis, occurred in the presence of ileum but not skeletal muscle or brain cytosols. This correlated with the stability of HxRh18-[3H]VCR complexes. In the presence of kidney cytosol, however, the molecular weight of 125I-tubulin remained unchanged, suggesting a different mechanism. Based upon data obtained, cytosols from normal tissues may be categorized into three classes: (a) those that formed stable complexes (brain); (b) those that formed unstable complexes but also destabilized preformed complex (ileum, kidney, liver, lung); and (c) tissues that formed unstable complexes but did not destabilized preformed complex (skeletal muscle, spleen, bone marrow, blood). The degree of instability of complexes formed in cytosols prepared from normal tissues appears to correlate with rapid loss of VCR from these tissues in vivo and hence may represent mechanism(s) for the selective action of this antineoplastic agent.

Type 1 (insulin-dependent) diabetes and a highly variable locus close to the insulin gene on chromosome 11.

A polymorphic DNA sequence in the 5'-flanking region of the human insulin gene was studied in relation to Type 1 (insulin-dependent) diabetes. In 141 Caucasoid subjects analysed by Southern blot hybridisation techniques, two major DNA insertions were observed: a Class 1 allele or a Class 3 allele. The Class 2 allele was not observed in this group of subjects. Genotype frequencies in a control population (n = 88) were: homozygous 1/1, 42%; heterozygous 1/3, 50%; and homozygous 3/3, 8%. Corresponding genotype frequencies in 53 Type 1 diabetic patients were 79%, 21% and 0%, respectively (p less than 0.0005 from chi 2 test). This confirms prevalence data reported by Bell et al. [16]. There appeared to be no coinheritance with HLA-DR3/DR4 related antigens, nor with autoimmune features. Analysis of 17 Type 1 diabetic pedigrees including 34 diabetic and 69 non-diabetic subjects did not demonstrate genetic linkage of these DNA inserts with diabetes, using an autosomal recessive, single locus model of inheritance.

Allelic variation adjacent to the human insulin and apolipoprotein C-II genes in different ethnic groups.

We have used DNA probes for the human insulin gene and apolipoprotein C-II (apo C-II) gene to determine the extent of allelic variation in different ethnic groups. The distribution of an apo C-II DNA polymorphism revealed by the restriction endonuclease Taq I showed no significant variation amongst racial groups; in contrast, an insulin gene-related DNA polymorphism showed marked variability. In Japanese, Chinese, and Asian Indian groups there was an increased frequency of homozygosity for the class 1 allele compared to Caucasian groups (P less than 0.001, P less than 0.01, and P less than 0.05, respectively). In Caucasian, Japanese, Chinese, and Asian Indian groups no class 2 allele was observed; but in the Negroid populations (African and West Indian) the class 2 allele frequencies were 0.23 and 0.25 respectively. Possible reasons for this variation in allele distribution are considered in relation to disease associations.

Spontaneous intercostal lung hernia.

Spontaneous intercostal lung herniae are infrequently described. Presented is a patient whose hernia arose after a vigorous cough. Treatment consisted of excision of the sac and repair. Demonstration of the defect roentgenographically and on physical examination is diagnostic. The defect can regress spontaneously; surgery is frequently curative, although techniques vary.

Insulin and apolipoprotein A-1/C-III gene polymorphisms relating to hypertriglyceridaemia and diabetes mellitus.

Two gene specific probes have been used to identify polymorphic DNA loci on chromosome 11 close to the insulin and apoprotein A-1 genes in a genetic analysis of hypertriglyceridaemic patients with and without co-existing diabetes. Of the 45 patients studied with both probes, 15 were diabetic of whom nine possessed class 3/3 insulin polymorphism genotypes, compared with none in the non-diabetic group (p less than 0.001; chi 2 test). In contrast, an uncommon apolipoprotein A-1 polymorphism was found to be equally distributed in the diabetic and the non-diabetic patients. No co-segregation of these two particular genetic polymorphisms was found in either patient group. The differing associations of the two disease-related polymorphism genotypes in patients with hypertriglyceridaemia with or without co-existing diabetes may possibly reflect differing aetiologies of the hyperlipidaemia.